Pyranosyl-RNA (‘p-RNA’): NMR and Molecular-Dynamics Study of the Duplex Formed by Self-pairing of Ribopyranosyl-(C-G-A-A-T-T-C-G)

The solution structure of the duplex formed by self-pairing of the p-RNA octamer β-D -ribopyranosyl-(2′→4′)-(CGAATTCG) was studied by NMR techniques and, independently, by molecular-dynamics calculations. The resonances of all non-exchanging protons, H-bearing C-atoms, P-atoms, and of most NH protons were assigned. Dihedral angle and distance constraints derived from coupling constants and NOESY spectra are consistent with a single dominant conformer and corroborate the main structural features predicted by qualitative conformational analysis. The duplex displays Watson-Crick pairing with antiparallel strand orientation. The dihedral angles β and ? in the phosphodiester linkages differ considerably from the idealized values. Model considerations indicate that these deviations from the idealized model allow better interstrand stacking and lessen unfavorable interactions in the backbone. The average base-pair axis forms an angle of ca. 40° with the backbone. The resulting interstrand π-π stacking between either two purines, or a purine and a pyrimidine, but not between two pyrimidines, constitutes a characteristic structural feature of the p-RNA duplex. A 1000-ps molecular-dynamics (MD) calculation with the AMBER force field resulted in an average structure of the same conformation type as derived by NMR. For the backbone torsion angle ?, dynamically averaged coupling constants from the MD calculation agree well with the experimental values, but for the angle β, a systematic difference of ca. 25° remains. The two base pairs at the ends of the duplex are calculated to be highly labile, which is consistent with the high exchange rate of the corresponding imino protons found by NMR.     

Solution structure of a peptide nucleic acid duplex from NMR data: features and limitations

This paper describes the results of a 1D and 2D NMR spectroscopy study of a palindromic 8-base pair PNA duplex GGCATGCC in H2O and H2O-D2O solutions. The (1)H NMR peaks have been assigned for most of the protons of the six central base pairs, as well as for several amide protons of the backbone. The resulting 36 interbase and base-backbone distance restraints were used together with Watson-Crick restraints to generate the PNA duplex structure in the course of 10 independent simulated annealing runs followed by restrained molecular dynamics (MD) simulations in explicit water. The resulting PNA structures correspond to a P-type helix with helical parameters close to those observed in the crystal structures of PNA. Based on the current limited number of restraints obtained from NMR spectra, alternative structures obtained by MD from starting PNA models based on DNA cannot be ruled out and are also discussed.     

Interpreting NMR data for beta-peptides using molecular dynamics simulations

NMR is one of the most used techniques to resolve structure of proteins and peptides in solution. However, inconsistencies may occur due to the fact that a polypeptide may adopt more than one conformation. Since the NOE distance bounds and (3)J-values used in such structure determination represent a nonlinear average over the total ensemble of conformers, imposition of NOE or (3)J-value restraints to obtain one unique conformation is not an appropriate procedure in such cases. Here, we show that unrestrained MD simulation of a solute in solution using a high-quality force field yields a conformational ensemble that is largely compatible with the experimental NMR data on the solute. Four 100 ns MD simulations of two forms of a nine-residue beta-peptide in methanol at two temperatures produced conformational ensembles that were used to interpret the NMR data on this molecule and resolve inconsistencies between the experimental NOEs. The protected and unprotected forms of the beta-peptide adopt predominantly a 12/10-helix in agreement with the qualitative interpretation of the NMR data. However, a particular NOE was not compatible with this helix indicating the presence of other conformations. The simulations showed that 3(14)()-helical structures were present in the ensemble of the unprotected form and that their presence correlates with the fulfillment of the particular NOE. Additionally, all inter-hydrogen distances were calculated to compare NOEs predicted by the simulations to the ones observed experimentally. The MD conformational ensembles allowed for a detailed and consistent interpretation of the experimental data and showed the small but specific conformational differences between the protected and unprotected forms of the peptide.     

Conserved patterns in backbone torsional changes allow for single base flipping from duplex DNA with minimal distortion of the double helix

Base flipping is a structural mechanism common to many DNA processing and repair enzymes. Changes in the local backbone torsions that occur during base flipping and the effect of environment on their behavior are of particular interest in understanding different base flipping mechanisms. In the present study, structures sampled during umbrella sampling molecular dynamics (MD) simulations of base flipping in aqueous and protein-bound environments, carried out with two different MD simulation strategies, are analyzed to find the most significant phosphodiester backbone distortions in the vicinity of the flipping base. Torsional sampling on the 5' side of the flipping base during flipping through the major groove shows similarities to the torsional sampling on the 3' side during flipping through the minor groove and vice versa. In differing environments, this behavior varies only marginally. These compensating torsional changes in the DNA backbone on 5' and 3' sides of the flipping base limit overall distortion of the DNA double helix during single base flipping. Rotameric intermediate states observed during base flipping are identified and postulated to be metastable states implicated in both large-scale structural changes and functional effects of chemical modifications in DNA.     

MD simulations of homomorphous PNA, DNA, and RNA single strands: characterization and comparison of conformations and dynamics

MD simulations of homomorphous single-stranded PNA, DNA, and RNA with the same base sequence have been performed in aqueous solvent. For each strand two separate simulations were performed starting from a (i) helical conformation and (ii) random coiled state. Comparisons of the simulations with the single-stranded helices (case i) show that the differences in the covalent nature of the backbones cause significant differences in the structural and dynamical properties of the strands. It is found that the PNA strand maintains its nice base-stacked initial helical structure throughout the 1.5-ns MD simulation at 300 K, while DNA/RNA show relatively larger fluctuations in the structures with a few local unstacking events during -ns MD simulation each. It seems that the weak physical coupling between the bases and the backbone in PNA causes a loss of correlation between the dynamics of the bases and the backbone compared to the DNA/RNA and helps maintain the base-stacked helical conformation. The global flexibility of a single-stranded PNA helix was also found to be lowest, while RNA appears to be the most flexible single-stranded helix. The sugar pucker of several nucleotides in single-stranded DNA and RNA was found to adopt both C2'-endo and C3'-endo conformations for significant times. This effect is more pronounced for single strands in completely coiled states. The simulations with single-stranded coils as the initial structure also indicate that a PNA can adopt a more compact globular structure, while DNA/RNA of the same size adopts a more extended coil structure. This allows even a short PNA in the coiled state to form a significantly stable nonsequentially base-stacked globular structure in solution. Due to the hydrophobic nature of the PNA backbone, it interacts with surrounding water rather weakly compared to DNA/RNA.     

Synthesis and DNA binding properties of novel benzo[b]isoquino[2,3-h]-naphthyridines

Several benzo[b]isoquino[2,3-h]-naphthyridines have been prepared via formal hetero-Diels Alder reaction of N-aryl imines as a key step. These compounds have different side chains at C-11, and a cis or trans configuration at the C-8a,C-14a ring junction. Binding constants for the interaction with oligonucleotides and polynucleotides were determined by UV absorption and melting experiments. NMR experiments (NOE) revealed that the cis isomers, showing a slightly folded structure, preferentially bind to the minor groove of AT-rich oligomers. In contrast, the trans isomers prefer the CG-rich sequences, leading to cap-complexes with the isoquinoline moiety stacked at the top of the double helix, in agreement with the flatter shape, and with a preference for the 3'-terminals, as found for camptothecins. Models of the complexes were built up by molecular dynamics (MD) calculations, by using the inter-proton distances derived from the NOE values. Cytotoxicity assays against human Ewing sarcoma cell lines RD-ES and CAD-ES1 were performed.     

Determination of aliphatic side-chain conformation using cross-correlated relaxation: application to an extraordinarily stable 2'-aminoethoxy-modified oligonucleotide triplex

The structural basis for the extraordinary stability of a triple-stranded oligonucleotide in which the third strand contains 2'-aminoethoxy-substituted riboses is investigated by NMR spectroscopy. The enhanced stability of the modified triplex in comparison to the unmodified DNA triplex of the same sequence can be attributed to strong interactions of the aminoethoxy groups of the third strand with the phosphate groups of the purine strand. In molecular dynamics calculations the aminoethoxy side chain was found to be rather flexible, allowing for the presence of hydrogen bonds between the aminoethoxy group of the third strand and two different phosphates of the backbone of the second strand. To investigate the conformational preference of the aminoethoxy side chain a new NMR method has been developed which relies on CH-CH dipolar-dipolar cross-correlated relaxation rates. The results indicate that the aminoethoxy side chains adopt mainly a gauche(+) conformation, for which only one of the two hydrogen bonds inferred by NMR and molecular dynamics simulations is possible. This demonstrates a highly specific interaction between the amino group of the third strand and one of the phosphate groups of the purine strand.     

Chiral Carbon Dots Mimicking Topoisomerase I To Mediate the Topological Rearrangement of Supercoiled DNA Enantioselectively

Nanomaterials with enzyme-mimetic activities are possible alternatives to natural enzymes. Mimicking enzymatic enantioselectivity remains a great challenge. Herein, we report that cysteine-derived chiral carbon dots (CDs) can mimic topoisomerase I to mediate topological rearrangement of supercoiled DNA enantioselectively. d -CDs can more effectively catalyze the topological transition of plasmid DNA from supercoiled to nicked open-circular configuration than l -CDs. Experiments suggest the underlying mechanism: d -CDs intercalatively bind with DNA double helix more strongly than l -CDs; the intercalative CDs can catalyze the production of hydroxyl radicals to cleave phosphate backbone in one strand of the double helix, leading to topological rearrangement of supercoiled DNA. Molecular dynamics (MD) simulation show that the stronger affinity for hydrogen-bond formation and hydrophobic interaction between d -cysteine and DNA than that of l -cysteine is the origin of enantioselectivity.     

Chiral Carbon Dots Mimicking Topoisomerase I To Mediate the Topological Rearrangement of Supercoiled DNA Enantioselectively

Nanomaterials with enzyme‐mimetic activities are possible alternatives to natural enzymes. Mimicking enzymatic enantioselectivity remains a great challenge. Herein, we report that cysteine‐derived chiral carbon dots (CDs) can mimic topoisomerase I to mediate topological rearrangement of supercoiled DNA enantioselectively. d ‐CDs can more effectively catalyze the topological transition of plasmid DNA from supercoiled to nicked open‐circular configuration than l ‐CDs. Experiments suggest the underlying mechanism: d ‐CDs intercalatively bind with DNA double helix more strongly than l ‐CDs; the intercalative CDs can catalyze the production of hydroxyl radicals to cleave phosphate backbone in one strand of the double helix, leading to topological rearrangement of supercoiled DNA. Molecular dynamics (MD) simulation show that the stronger affinity for hydrogen‐bond formation and hydrophobic interaction between d ‐cysteine and DNA than that of l ‐cysteine is the origin of enantioselectivity.     

Propagation of melting cooperativity along the phosphodiester backbone of DNA

The role of the DNA phosphodiester backbone in the transfer of melting cooperativity between two helical domains was experimentally addressed with a helix-bulge-helix DNA model, in which the bulge consisted of a varying number of either conformationally flexible propanediol or conformationally constrained bicyclic anucleosidic phosphodiester backbone units. We found that structural communication between two double helical domains is transferred along the DNA backbone over the equivalent of ca. 12-20 backbone units, depending on whether there is a symmetric or asymmetric distribution of the anucleosidic units on both strands. We observed that extension of anucleosidic units on one strand only suffices to disrupt cooperativity transfer in a similar way as if extension occurs on both strands, indicating that the length of the longest anucleosidic inset determines cooperativity transfer. Furthermore, conformational rigidity of the sugar unit increases the distance of coopertivity transfer along the phosphodiester backbone. This is especially the case when the units are asymmetrically distributed in both strands.     

DNA recognition by the anthracycline antibiotic respinomycin D: NMR structure of the intercalation complex with d(AGACGTCT)2

Respinomycin D is a member of the anthracycline family of antitumour antibiotics that interact with double stranded DNA through intercalation. The clinical agents daunomycin and doxorubicin are the most well-studied of this class but have a relatively simple molecular architecture in which the pendant daunosamine sugar resides in the DNA minor groove. Respinomycin D, which belongs to the nogalamycin group of anthracyclines, possesses additional sugar residues at either end of the aglycone chromophore that modulate the biological activity but whose role in molecular recognition is unknown. We report the NMR structure of the respinomycin D-d(AGACGTCT)2 complex in solution derived from NOE restraints and molecular dynamics simulations. We show that the drug threads through the DNA double helix forming stabilising interactions in both the major and minor groove, the latter through a different binding geometry to that previously reported. The bicycloaminoglucose sugar resides in the major groove and makes specific contacts with guanine at the 5'-CpG intercalation site, however, the disaccharide attached at the C4 position plays little part in drug binding and DNA recognition and is largely solvent exposed.     

RNA unrestrained molecular dynamics ensemble improves agreement with experimental NMR data compared to single static structure: a test case

Nuclear magnetic resonance (NMR) provides structural and dynamic information reflecting an average, often non-linear, of multiple solution-state conformations. Therefore, a single optimized structure derived from NMR refinement may be misleading if the NMR data actually result from averaging of distinct conformers. It is hypothesized that a conformational ensemble generated by a valid molecular dynamics (MD) simulation should be able to improve agreement with the NMR data set compared with the single optimized starting structure. Using a model system consisting of two sequence-related self-complementary ribonucleotide octamers for which NMR data was available, 0.3 ns particle mesh Ewald MD simulations were performed in the AMBER force field in the presence of explicit water and counterions. Agreement of the averaged properties of the molecular dynamics ensembles with NMR data such as homonuclear proton nuclear Overhauser effect (NOE)-based distance constraints, homonuclear proton and heteronuclear ¹H–³¹P coupling constant (J) data, and qualitative NMR information on hydrogen bond occupancy, was systematically assessed. Despite the short length of the simulation, the ensemble generated from it agreed with the NMR experimental constraints more completely than the single optimized NMR structure. This suggests that short unrestrained MD simulations may be of utility in interpreting NMR results. As expected, a 0.5 ns simulation utilizing a distance dependent dielectric did not improve agreement with the NMR data, consistent with its inferior exploration of conformational space as assessed by 2-D RMSD plots. Thus, ability to rapidly improve agreement with NMR constraints may be a sensitive diagnostic of the MD methods themselves.     

Complexation between tert-butyl ketones and beta-cyclodextrin. Structural study by NMR and MD simulations

A structural study (NMR and MD) of the complexation between tert-butyl ketones and beta-cyclodextrin has been performed. A priority order for the alkyl and phenyl groups composing the ketones has been determined based on association constants: Ph- > C(6)H(11)- = t-Bu- > Bu-, Pr-, Me-. Geometries for the complexes are proposed based on NOE values and on the MD simulations. Bimodal complexation occurs in all the compounds studied.     

Structure of a cyclodextrin functionalized anionic clay: XRD analysis, spectroscopy, and computer simulations

Carboxy-methyl beta-cyclodextrin (CMCD) cavities have been intercalated within the galleries of anionic clay, Mg-Al layered double hydroxide (LDH). The cyclodextrin functionalized LDH has been reported to adsorb neutral and nonpolar guest molecules. X-ray diffraction, IR, and Raman vibrational spectroscopy and (13)C CPMAS NMR have been used to characterize the confined CMCD molecules, whereas molecular dynamics simulations have been used to probe the interlayer arrangement and orientation of the intercalated species. Spectroscopic measurements as well as MD simulations show that there is no significant change in the geometry of the CMCD cavity on intercalation. Within the galleries of the anionic clay, the CMCD anions are arranged as bilayers with the carboxy methyl substituents, located at the narrower opening of the bucket-like cyclodextrin toroid, anchored to the LDH sheet. This arrangement leaves the wider opening of the CMCD anion facing away from the layers allowing the interior of the cyclodextrin cavity to be accessible to guest molecules. Finally, the hydrophobicity of the anchored cyclodextrin cavity has been characterized using fluorescence from pyrene included within it.     

Simulation of adsorption of DNA on carbon nanotubes

We report molecular dynamics simulations of DNA adsorption on a single-walled carbon nanotube (SWNT) in an aqueous environment. We have modeled a DNA segment with 12 base pairs (Dickerson dodecamer) and a (8,8) SWNT in water, with counterions to maintain total charge neutrality. Simulations show that DNA binds to the external surface of an uncharged or positively charged SWNT on a time scale of a few hundred picoseconds. The hydrophobic end groups of DNA are attracted to the hydrophobic SWNT surface of uncharged SWNTs, while the hydrophilic backbone of DNA does not bind to the uncharged SWNT. The binding mode of DNA to charged SWNTs is qualitatively different from uncharged SWNTs. The phosphodiester groups of the DNA backbone are attracted to a positively charged SWNT surface while DNA does not adsorb on negatively charged SWNTs. There is no evidence for canonical double-stranded DNA wrapping around either charged or uncharged SWNTs on the very short time scales of the simulations. The adsorption process appears to have negligible effect on the internal stacking structure of the DNA molecule but significantly affects the A to B form conversion of A-DNA. The adsorption of A-DNA onto an uncharged SWNT inhibits the complete relaxation of A-DNA to B-DNA within the time scale of the simulations. In contrast, binding of the A-DNA onto a positively charged SWNT may promote slightly the A to B conversion.     

Structural characterization and biological evaluation of small interfering RNAs containing cyclohexenyl nucleosides

CeNA is an oligonucleotide where the (deoxy)ribose sugars have been replaced by cyclohexenyl moieties. We have determined the NMR structure of a CeNA:RNA duplex and have modeled this duplex in the crystal structure of a PIWI protein. An N puckering of the ribose nucleosides, a 2H3 conformation of the cyclohexenyl nucleosides, and an A-like helix conformation of the backbone, which deviates from the standard A-type helix by a larger twist and a smaller slide, are observed. The model of the CeNA:RNA duplex bound to the PIWI protein does not show major differences in the interaction of the guide CeNA with the protein when compared with dsRNA, suggesting that CeNA modified oligonucleotides might be useful as siRNAs. Incorporation of one or two CeNA units in the sense or antisense strands of dsRNA led to similar or enhanced activity compared to unmodified siRNAs. This was tested by targeting inhibition of expression of the MDR1 gene with accompanying changes in P-glycoprotein expression, drug transport, and drug resistance.