Studies on the cross stand hydrogen bonds in DNA double helices.

A systematic analysis has been carried out to examine all the stereochemically possible bifurcated hydrogen bonds including those of cross strand type between propeller twisted base pairs in DNA double helices by stereochemical considerations involving base pairs alone and by molecular mechanics studies on dimer and trimer duplexes. The results show that there are limited number of combinations of adjacent base pairs that would facilitate bifurcated cross-strand hydrogen bond (CSH). B-type helices concomitant with negative propeller twist seem to be more favored for the occurrence of CSH than canonical A-type helices because of slide in the latter. The results also demonstrate that helices with appropriate sequences may possess continuous run of these propeller twist driven cross strand hydrogen bonds indicating that they may in fact be considered as yet another general structural feature of DNA helices.     

Studies On The Cross Strand Hydrogen Bonds In DNA Double Helices

Abstract

A systematic analysis has been carried out to examine all the stereochemically possible bifurcated hydrogen bonds including those of cross strand type between propeller twisted base pairs in DNA double helices by stereochemical considerations involving base pairs alone and by molecular mechanics studies on dimer and trimer duplexes. The results show that there are limited number of combinations of adjacent base pairs that would facilitate bifurcated cross- strand hydrogen bond (CSH). B-type helices concomitant with negative propeller twist seem to be more favored for the occurrence of CSH than canonical A-type helices because of slide in the latter. The results also demonstrate that helices with appropriate sequences may possess continuous run of these propeller twist driven cross strand hydrogen bonds indicating that they may infact be considered as yet another general structural feature of DNA helices.     

Comparison of interproton distances in DNA models with nuclear Overhauser enhancement data

The conformational flexibility inherent in the polynucleotide chain plays an important role in deciding its three-dimensonal structure and enables it to undergo structural transitions in order to fulfil all its functions. Following certain stereochemical guidelines, both right and left handed double-helical models have been built in our laboratory and they are in reasonably good agreement with the fibre patterns for various polymorphous forms of DNA. Recently, nuclear magnetic resonance spectroscopy has become an important technique for studying the solution conformation and polymorphism of nucleic acids. Several workers have used 1_H nuclear magnetic resonance nuclear Overhauser enhancement measurements to estimate the interproton distances for the various DNA oligomers and compared them with the-interproton distances for particular models of A and B form DNA. In some cases the solution conformation does not seem to fit either of these models. We have been studying various models for DNA with a view to exploring the full conformational space allowed for nucleic acid polymers. In this paper, the interproton distances calculated for the different stereochemically feasible models of DNA are presented and they are compared and correlated against those obtained from¹H nuclear magnetic resonance nuclear Overhauser enhancement measurements of various nucleic acid oligomers.     

Stereochemical Effects of Methylphosphonate in B- and Z-DNA Helices: Variation in Hydrophobicity and Effective Widths of Grooves

Abstract

Stereochemical effects of methylphosphonate (MP) in B-DNA and Z-DNA duplexes are studied through molecular mechanics approach. Duplexes of different lengths, tetramers, hexamers, dodecamers are examined to assess the interstrand and intrastrand electrostatic effects due to MPs vis-a-vis phosphates. A variety of models which include duplexes with alternating S-MP and R-MP, alternating phosphate and MP and, duplexes posessing MPs in only one of the strands, are examined by considering both the S- and R-stereoisomers. Majority of the calculations are performed with CG sequences to delineate factors responsible for the stability of B- and Z-DNA as well as B × Z-DNA transition under nonionic conditions. The results show that both B- and Z-DNA duplexes are energetically favoured in the presence of MP due to overwhelming reduction in intrastrand as well as interstrand electrostatic repulsive interactions. The effect is distinct in oligomers longer than tetramers. Comparison of energetics of MP B- and Z-DNA duplexes suggests that an oligodeoxynucleotide such as d(CG)₆ with all phosphates replaced by MPs may favour equally both B- and Z-DNA conformations. The analysis further provides an estimate of electrostatic interactions, operating at the grooves under a variety of conditions. Several specific and localised effects due to S-MP and R-MP are seen at CG and GC steps in various B-DNA and Z-DNA models. S-MP in B- DNA reduces the effective major groove width by nearly 3 Å hence denying access to the functional groups of endonucleases thereby enhancing the resistance of MP-DNA to enzymatic digestion. Further, methyl groups of MP render the surface of the DNA helix to be significantly hydrophobic which may explain higher permeability of MP-DNA in membranes as well as its less soluble nature in aqueous media.     

Structural basis for the unusual properties of 2',5' nucleic acids and their complexes with RNA and DNA

To provide insights into the unusual properties of 2',5' nucleic acids (iso nucleic acids), that includes their rejection by Nature as information molecules, modeling studies have been carried out to examine if they indeed possess the stereochemical ability to form helical duplexes and triplexes, just as their 3',5' linked constitutional isomers. The results show that the formation of helical duplexes with 2',5' linkages demands a mandatory displacement of the Watson and Crick base pairs from the helical axis, as a direct consequence of the lateral shift of the sugar-phosphate backbone from the periphery towards the interior of the helix. Thus, both duplexes and triplexes formed with a 2',5'-sugar-phosphate backbone possess this intrinsic trait, manifested normally only in A type duplexes of DNA and RNA. It was found that only a 10-fold symmetric parallel triplex with isomorphous T.AT triplets is stereochemically favorable for isoDNA with 'extended' nucleotide repeats, unlike the 12-fold symmetric triplex favored by DNA. The wider nature of a 12-fold triplex, concomitant with mandatory slide requirement for helix formation in isoDNA, demands even larger displacement, especially with 'extended' nucleotide structural repeats, thereby violating symmetry. However, a symmetric triplex possessing higher twist, can be naturally formed for isoDNA with a 'compact' nucleotide repeat. Two nanosecond molecular dynamics simulation of a 2',5'-B DNA duplex, formed with an intrinsic base pair displacement of -3.3 A, does not seem to favor a total transition to a typical A type duplex, although enhanced slide, X-displacement, decrease in helical rise and narrowing of the major groove during simulation seem to indicate a trend. Modeling of the interaction between the chimeric isoDNA.RNA duplex and E. coli RNase H has provided a structural basis for the inhibitory action of the enzyme. Interaction of residues Gln 80, Trp 81, Asn 16 and Lys 99, of E. coli RNase H with DNA of the DNA.RNA hybrid, are lost when the DNA backbone is replaced by isoDNA. Based on modeling and experimental observations, it is argued that 2',5' nucleic acids possess restricted conformational flexibility for helical polymorphism. The inability of isoDNA to favor the biologically relevant B form duplex and the associated topological inadequacies related to nucleic acid compaction and interactions with regulatory proteins may be some of the factors that might have led to the rejection of 2',5' links.     

Sequence-dependent molecular conformation of polynucleotides: right and left-handed helices

Based upon a stereochemical guideline, two topologically distinct types of helicalduplexes have been deduced for a polynucleotide duplex with alternating purine pyrimidine sequence (PAPP): (a) right-handed uniform (RU) helix and (b) left-handed zig-zag (LZ) helix. Both structures have trinucleoside diphosphate as the basic unit wherein the purine pyrimidine fragment has a different conformation from the pyrimidine-purine fragment. Thus, RU and LZ helices represent two different classes of sequence-dependent molecular conformations for PAPP. The conformationalf eatures of an RU helix of PAPP in B-form and three LZ-helices for B-, D- and Z-forms are discussed.     

NMR solution structures of [d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2] and its unmodified control.

We present the high-resolution solution structures of a self-complementary DNA decamer duplex featuring a single alpha-anomeric nucleotide per strand encompassed by a set of 3'-3' and 5'-5' phosphodiester linkages, d(GCGAAT-3'-3'-alphaT-5'-5'-CGC)2, alphaT, and its unmodified control, d(GCGAATTCGC)2, obtained by restrained molecular dynamics. Interproton distance and deoxyribose ring torsion angle restraints were deduced from homonuclear NOESY and DQF-COSY data, respectively. For both the control and alphaT duplexes, excellent global convergence was observed from two different (A- and B-) starting models. The final average structures of the two duplexes are highly homologous, and overall possess the traits characteristic of right-handed B-DNA duplexes. However, localized differences between the two structures stem from the enhanced conformational exchange in the deoxyribose ring of the cytidine following the 5'-5' linkage, the C3'- exo pseudorotation phase angle of the alpha-nucleotide, and unusual backbone torsions in the 3'-3' and 5'-5' phosphodiester linkages. The structural data reported here are relevant to the design of antisense therapeutics comprised of these modifications.     

The stereochemical complementarity of DNA and reproductive steroid hormones correlates with biological activity

Modeling studies revealed that progesterone, testosterone, and estradiol are stereochemically complementary to the cavity formed between base pairs in the DNA sequence, 5'-dTdG-3' X 5'-dCdA-3'. Each steroid aligned precisely with the topography of the cavity and formed 2 stereospecific hydrogen bonds linking phosphate oxygens on adjacent DNA strands. Hydrogen bonding donor-acceptor relationships were different for each hormone. The remarkable stereochemical specificity of the hormone-DNA complexes was demonstrated by the lack of complementarity of steroid enantiomers and steroid analogs having alternate ring systems and/or changes in the position of functional groups. Fit of molecules into DNA in the manner of the parent hormone correlated with biological activity. Antagonists also fit into the cavity but differed from agonists in their hydrogen bonding linkages to DNA and/or extended out of the cavity beyond the helix. Unlike flat intercalating agents which form stable complexes with DNA, wedge shaped steroids may thus be capable of forming reversible sequence-specific complexes with DNA. We conclude that the stereochemistry of DNA can be used to predict hormonal activity.     

An accurate method for determining the helical repeat of DNA in solution reveals differences to the crystal structures of two B-DNA decamers.

Many DNA sequences have been studied by X-ray crystallography with the goal of deciphering a sequence-structure code. We have determined the helical repeats of two B-type DNA decamers in solution employing an electrophoretic method based on phasing of bent segments. The decamers contain recognition sites for the dcm methyltransferase and for the restriction nuclease NarI with a mutational hotspot. Their helical repeats are 10.59(+/- 0.05) bp and 10.52(+/- 0.03) bp, respectively, whereas crystallographic analysis yielded 10.0 bp in the solid state. This difference is greater than that for the transition between B- and A-type DNA in solution. Thus, reliable information about the polymorphism of DNA in solution must be based on both X-ray and solution data. We describe a generally applicable approach to accurately determine helical repeats of small DNA duplexes in solution.     

4-hydroxyequilenin-adenine lesions in DNA duplexes: stereochemistry, damage site, and structure

The equine estrogens, equilin and equilenin, are major components of the drug Premarin, the most widely used formula for hormone replacement therapy. The derivative 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react in vitro and in vivo with cytosine and adenine in DNA. Unique cyclic adducts containing the same bicyclo[3.3.1]nonane-type connection ring are produced. Each base adduct has four stereoisomers. In order to elucidate the structural effects of A versus C modification, we have carried out molecular dynamics simulations of the stereoisomeric 4-OHEN-A adducts in DNA 11-mer duplexes and compared results with an earlier study of the C adducts (Ding, S., Shapiro, R., Geacintov, N.E., and Broyde, S. (2005) Equilenin-Derived DNA Adducts to Cytosine in DNA Duplexes: Structures and Thermodynamics, Biochemistry 44, 14565-14576). Similar stereochemical principles govern the orientations in DNA duplexes of the 4-OHEN-A adducts as for the analogous C adducts, with opposite orientations of the equilenin rings in stereoisomeric pairs of adducts characterized by near-mirror image circular dichroism (CD) spectra. However, the larger purine adducts have unique structural properties in the duplexes that distinguish their characteristics from those of the pyrimidine adducts. Significant differences are observed in terms of hydrogen bonding, stacking, bending, groove dimensions, solvent exposure, and hydrophobic interactions; also, each of the four stereoisomeric 4-OHEN-A adducts exhibit distinct structural features. Each base adduct and stereoisomer distorts the structure of the DNA duplex differently. These characteristics may manifest themselves in terms of differential nucleotide excision repair susceptibilities and mutagenic activities of the 4-OHEN-A and C adducts.     

A mixed-alpha,beta miniprotein stereochemically reprogrammed to high-binding affinity for acetylcholine

The protein-structure space is limited to L configuration in the asymmetric alpha-amino acid structures; the function space, on other hand, seems limitless because of the chemical diversity in the amino acid side chain structures. The chemical diversity in side chain structure may be multiplied beneficially with the stereochemical diversity in main chain structure; thus, de novo protein design may be explored for customizing molecular structures stereochemically and molecular functions chemically. Illustrating de novo design in the structure space of L and D alphabet, canonical all-beta folds of poly-L structure were reprogrammed as bracelet, boat, and canoe-shaped molecules-the "boat" as a receptor-like pocket and the "canoe" as a metal-ion receptor-simply by mutating specific L-amino acid residues to the corresponding D stereochemical structure. Demonstrating customization of molecular shape stereochemically and function chemically, a 15-residue mixed-alpha, beta miniprotein of canonical poly-L structure is now reprogrammed stereochemically as a cup-shaped receptor for acetylcholine via cation-pi interaction with a triad of aromatic side chains placed in mimicry of the acetylcholine-receptor sites both natural and artificial. Evidence from CD, fluorescence, NMR, DSC, ITC, MD, and molecular-docking studies is presented to show that a rationally designed 15-residue mixed-L, D peptide is a cooperatively ordered molecular fold in the stereochemically specified molecular morphology, submicromolar in affinity of acetylcholine and thus an acetylcholine receptor exceptionally small and simple. .     

Stereochemistry-dependent bending in oligonucleotide duplexes induced by site-specific covalent benzo[a]pyrene diol epoxide-guanine lesions.

The apparent persistence length of enzymatically linearized pIBI30 plasmid DNA molecules approximately 2300 bp long, as measured by a hydrodynamic linear flow dichroism method, is markedly decreased after covalent binding of the highly tumorigenic benzo[a]pyrene metabolite 7R,8S-dihydroxy-9S,10R-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+)-anti-BPDE]. In striking contrast, the binding of the non-tumorigenic, mirror-image 7S,8R,9R,10S enantiomer [(-)-anti-BPDE] to DNA has no measurable effect on its alignment in hydrodynamic flow gradients (< or = 2.2% of the DNA bases modified). In order to relate this effect to BPDE-nucleotide lesions of defined stereochemistry, the bending induced by site-specifically placed and stereochemically defined (+)- and (-)-anti-BPDE-N2-dG lesions in an 11mer deoxyoligonucleotide duplex was studied by ligation and gel electrophoresis methods. Out of the four stereochemically isomeric anti-BPDE-N2-deoxyguanosyl (dG) adducts with either (+)-trans, (-)-trans, (+)-cis, and (-)-cis adduct stereochemistry, only the (+)-trans adduct gives rise to prominent bends or flexible hinge joints in the modified oligonucleotide duplexes. Since both anti-BPDE enantiomers are known to bind preferentially to dG (> or = 85%), these observations can account for the differences in persistence lengths of DNA modified with either (+)-anti-BPDE or the chiral (-)-anti-BPDE isomer.     

Intramolecular synapsis of duplex DNA by vaccinia topoisomerase.

Complexes formed by vaccinia topoisomerase I on plasmid DNA were visualized by electron microscopy. The enzyme formed intramolecular loop structures in which non-contiguous DNA segments were synapsed within filamentous protein stems. At high enzyme concentrations the DNA appeared to be zipped up within the protein filaments such that the duplex was folded back on itself. Formation of loops and filaments was also observed with an active site mutant, Topo-Phe274. Binding of Topo-Phe274 to relaxed DNA circles in solution introduced torsional strain, which, after relaxation by catalytic amounts of wild-type topo-isomerase, resulted in acquisition of negative supercoils. We surmise that the topoisomerase-DNA complex is a plectonemic supercoil in which the two duplexes encompassed by the protein filaments are interwound in a right handed helix. We suggest that topoisomerase-mediated DNA synapsis plays a role in viral recombination and in packaging of the 200 kbp vaccinia genome during virus assembly.     

Synthesis and stereochemistry of the terminal spiroketal domain of the phosphatase inhibitor dinophysistoxin-2

An expedient synthesis of both axially and equatorially C35 methyl substituted spiroketals representing the C28-C38 domain of the potent and selective protein serine/threonine phosphatase inhibitor dinophysistoxin-2 (DTX-2) was developed to enable comparative stereochemical analyses and a stereochemically correct total synthesis of DTX-2. Comparison of proton and carbon NMR data of the synthetic diastereomers with those published for DTX-2 indicates that DTX-2 possesses the (30S *,34R *,35S *)-relative configuration with an axial C35 methyl substituent.     

The influence of DNA sequence on terbium (III) fluorescence enhancement by DNA

The synthetic DNA duplexes, poly(dA-dC):poly(dG-dT), poly(dG):poly(dC), poly(dG-dC):poly(dG-dC), and poly(dG-m5dC):poly(dG-m5dC), were analyzed as double- and single-strand polymers for the ability to enhance terbium fluorescence. Using conditions which limited the enhancement of Tb3+ fluorescence to that from DNA-guanosines, our results showed that (a) guanosines in single-strand DNA enhanced terbium fluorescence equally well irrespective of the primary sequence surrounding them, and (b) guanosines in either left- (Z-form) or right- (B-form) handed double helixes failed to enhance terbium fluorescence.     

STRUCTURAL,DYNAMIC, AND ENZYMATIC PROPERTIES OF MIXED α/β-OLIGONUCLEOTIDES CONTAINING POLARITY REVERSALS

We employ NMR structure determination, thermodynamics, and enzymatics to uncover the structural, thermodynamic and enzymatic properties of α/β-ODNs containing 3′-3′ and 5′-5′ linkages. RNase H studies show that α/β-gapmers that are designed to target erbB-2 efficiently elicit RNase H activity. NMR structures of DNA · DNA and DNA · RNA duplexes reveal that single α-anomeric residues fit well into either duplex, but alter the dynamic properties of the backbone and deoxyriboses as well as the topology of the minor groove in the DNA · RNA hybrid.     

Transcription regulation by steroid hormones: a computer simulation study.

Three-dimensional structures of complexes of 66 amino acid-DNA binding domains of human progesterone (hPR), estrogen (hER) and glucocorticoid (hGR) receptors (proteins), with ten base pair DNA duplexes: d(AGGTCATGCT).d(AGCATGACCT) and d(AGAACATGCT).d(AGCATGTTCT) were obtained using computer modeling and molecular mechanics techniques. Cartesian coordinates for the proteins were obtained from: 1) structural data of hER and hGR by NMR spectroscopy; 2) steric constraints imposed by tetrahedral coordination of the zinc ion to Cys residues, and 3) energy minimization in torsional and cartesian space. The proteins were made to interact with DNA (in B-form) in major groove through alpha-helical linker between the two zinc fingers. The geometry of the complexes was obtained by allowing them to slide, glide, penetrate in to and out of the groove, and to rotate about the helical axis. The complexes were energy minimized. Also maximized was the number of H-bonds between proteins and DNA. The complex structures were refined by molecular mechanics using AMBER 3.0. Structural parameters of DNA were analyzed in each complex and compared with those of native DNA optimized separately. The stereochemical differences of the complexes are discussed.