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.     

Hole transfer rates in A-form DNA/2'-OMeRNA hybrid

The hole transfer rates in the DNA/DNA B-form duplex and DNA/2'-OMeRNA A-form duplex were measured which occurred in the time range of approximately 100 micros. The hole transfer rates in the A-form duplexes were slower and more strongly dependent on the temperature compared to those in the B-form duplexes, suggesting that the A-form is more rigid than the B-form duplex in this time scale.     

DNA-mediated charge transport requires conformational motion of the DNA bases: elimination of charge transport in rigid glasses at 77 K

We have proposed that DNA-mediated charge transport (CT) is gated by base motions, with only certain base conformations being CT-active; a CT-active conformation can be described as a domain, a transiently extended pi-orbital defined dynamically by DNA sequence. Here, to explore these CT-active conformations, we examine the yield of base-base CT between photoexcited 2-aminopurine (Ap*) and guanine in DNA in rigid LiCl glasses at 77 K, where conformational rearrangement is effectively eliminated. Duplex DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)nG (n = 0-4). The yield of CT was monitored through fluorescence quenching of Ap* by G. We find, first, that the emission intensity of Ap* in all DNA duplexes increases dramatically upon cooling and becomes comparable to free Ap*. This indicates that all quenching of Ap* in duplex DNA is a dynamic process that requires conformational motion of the DNA bases. Second, DNA-mediated CT between Ap* and G is not observed at 77 K; rather than hindering the ability of DNA to transport charge, conformational motion is required. Moreover, the lack of DNA-mediated CT at 77 K, even through the shortest bridge, suggests that the static structures adopted upon cooling do not represent optimum CT-active conformations. These observations are consistent with our model of conformationally gated CT. Through conformational motion of the DNA bases, CT-active domains form and break-up transiently, both facilitating and limiting CT.     

Direct chemical evidence for charge transfer between photoexcited 2-aminopurine and guanine in duplex DNA

Photoexcited 2-aminopurine (Ap*) is extensively exploited as a fluorescent base analogue in the study of DNA structure and dynamics. Quenching of Ap* in DNA is often attributed to stacking interactions between Ap* and DNA bases, despite compelling evidence indicating that charge transfer (CT) between Ap* and DNA bases contributes to quenching. Here we present direct chemical evidence that Ap* undergoes CT with guanine residues in duplex DNA, generating oxidative damage at a distance. Irradiation of Ap in DNA containing the modified guanine, cyclopropylguanosine (CPG), initiates hole transfer from Ap* followed by rapid ring opening of the CPG radical cation. Ring opening accelerates hole trapping to a much shorter time regime than for guanine radicals in DNA; consequently, trapping effectively competes with back electron transfer (BET) leading to permanent CT chemistry. Significantly, BET remains competitive, even with this much faster trapping reaction, consistent with measured kinetics of DNA-mediated CT. The distance dependence of BET is sharper than that of forward CT, leading to an inverted dependence of product yield on distance; at short distances product yield is inhibited by BET, while at longer distances trapping dominates, leading to permanent products. The distance dependence of product yield is distinct from forward CT, or charge injection. As with photoinduced charge transfer in other chemical and biological systems, rapid kinetics for charge injection into DNA need not be associated with a high yield of DNA damage products.     

The structure of a mixed LNA/DNA:RNA duplex is driven by conformational coupling between LNA and deoxyribose residues as determined from 13C relaxation measurements

A study of the internal dynamics of an LNA/DNA:RNA duplex has been performed to further characterize the conformational changes associated with the incorporation of locked nucleic acid (LNA) nucleotides in a DNA:RNA duplex. In general, it was demonstrated that the LNA/DNA:RNA duplex has a very high degree of order compared to dsDNA and dsRNA duplexes. The order parameters of the aromatic carbon atoms in the LNA/DNA strand are uniformly high, whereas a sharp drop in the degree of order was seen in the RNA strand in the beginning of the AUAU stretch in the middle of the strand. This can be related to a return to normal dsRNA dynamics for the central A:U base pair. The high order of the heteroduplex is consistent with preorganization of the chimera strand for an A-form duplex conformation. These results partly explain the dramatic increase in T(m) of the chimeric heteroduplex over dsDNA and DNA:RNA hybrids of the same sequence.     

Input-dependent induction of oligonucleotide structural motifs for performing molecular logic

The K(+)-H(+)-triggered structural conversion of multiple nucleic acid helices involving duplexes, triplexes, G-quadruplexes, and i-motifs is studied by gel electrophoresis, circular dichroism, and thermal denaturation. We employ the structural interconversions for perfoming molecular logic operations, as verified by fluorimetry and colorimetry. Short G-rich and C-rich cDNA and RNA single strands are hybridized to produce four A-form and B-form duplexes. Addition of K(+) triggers the unwinding of the duplexes by inducing the folding of G-rich strands into DNA- or RNA G-quadruplex mono- and multimers, respectively. We found a decrease in pH to have different consequences on the resulting structural output, depending on whether the C-rich strand is DNA or RNA: while the protonated C-rich DNA strand folds into at least two isomers of a stable i-motif structure, the protonated C-rich RNA strand binds a DNA/RNA hybrid duplex to form a Y·RY parallel triplex. When using K(+) and H(+) as external stimuli, or inputs, and the induced G-quadruplexes as reporters, these structural interconversions of nucleic acid helices can be employed for performing logic-gate operations. The signaling mode for detecting these conversions relies on complex formation between DNA or RNA G-quadruplexes (G4) and the cofactor hemin. The G4/hemin complexes catalyze the H(2)O(2)-mediated oxidation of peroxidase substrates, resulting in a fluorescence or color change. Depending on the nature of the respective peroxidase substrate, distinct output signals can be generated, allowing one to operate multiple logic gates such as NOR, INH, or AND.     

Synthesis and properties of nucleic acid analogues consisting of a benzene-phosphate backbone

[Chemical reaction: See text] The synthesis and properties of a nucleic acid analogue consisting of a benzene-phosphate backbone are described. The building blocks of the nucleic acid analogue are composed of bis(hydroxymethyl)benzene residues connected to nucleobases via the biaryl-like axis. Stabilities of the duplexes were studied by thermal denaturation. It was found that the thermal stabilities of the duplexes composed of the benzene-phosphate backbone are highly dependent on their sequences. The duplexes with the benzene-phosphate backbone comprised of the mixed sequences were thermally less stable than the natural DNA duplexes, whereas that composed of the homopyrimidine and homopurine sequences was thermally and thermodynamically more stable than the corresponding natural DNA duplex. It was suggested that the analogues more efficiently stabilize the duplexes in a B-form duplex rather than in an A-form duplex. Thus, the duplexes consisting of the benzene-phosphate backbone, especially composed of the homopyrimidine and homopurine sequences, may offer a novel structural motif useful for developing novel materials applicable in the fields of bio- and nanotechnologies.     

Duplex structure of a minimal nucleic acid

The crystal structure of an 8-mer (S)-GNA duplex is presented. As a tool for phasing, the anomalous diffraction of two copper(II) ions within two artificial metallo-base pairs was employed. The duplex structure confirms a canonical Watson-Crick base pairing scheme of GNA with antiparallel strands. The duplex secondary structure is distinct from canonical A- and B-form nucleic acids and can be described as a right-handed helical ribbon wrapped around the helix axis, resulting in a large hollow core. Most intriguingly, neighboring base pairs slide strongly against each other, resulting in extensive interstrand base-base hydrophobic interactions along with unusual hydrophobic intrastrand interactions of nucleobases with their backbone. These results reveal how a minimal nucleic acid backbone can support highly stable Watson-Crick-like duplex formation.     

Atomic detail investigation of the structure and dynamics of DNA.RNA hybrids: a molecular dynamics study

DNA.RNA hybrid duplexes are biologically important molecules and are shown to have potential therapeutic properties. To investigate the relationship between structures, energetics, solvation and RNase H activity of hybrid duplexes in comparison with pure DNA and RNA duplexes, a molecular dynamics study using the CHARMM27 force field was undertaken. The structural properties of all four nucleic acids considered are in very good agreement with the experimental data. The backbone dihedral angles and the puckering of the (deoxy)ribose indicate that the purine rich strands retain their A-/B-like properties but the pyrimidine rich DNA strand undergoes A-B conformational transitions. The minor groove widths of the hybrid structures are narrower than those in the RNA duplex, a requirement for RNase H binding. In addition, sampling of noncanonical phosphodiester backbone dihedrals by the DNA strands, differential solvation properties and helical properties, most notably rise, are suggested to contribute to hybrids being RNase H substrates. Differential RNase H activity toward hybrids containing purine versus pyrimidine rich RNA strands is suggested to be due to sampling of values of the phosphodiester backbone dihedrals in the DNA strands. Notably, the present results indicate that hybrids have decreased flexibility as compared to RNA, in contrast to previous reports.     

Assignment and NOE analysis of 2'-hydroxyl protons in RNA: implications for stabilization of RNA A-form duplexes

The ribose 2'-OH hydroxyl group distinguishes RNA from DNA. The 2'-OH hydroxyl protons are responsible for differences in conformation, hydration, and thermodynamic stability of RNA and DNA oligonucleotides. Additionally, the 2'-OH group plays a central role in RNA catalysis. This important group lies in the shallow groove of RNA, where it is involved in a network of hydrogen bonds with water molecules stabilizing RNA A-form duplexes. Structural and dynamical information on 2'-OH hydroxyl protons is essential to understand their respective roles. Here we report the 2'-OH hydroxyl proton assignments for a 30mer RNA, the HIV-2 transactivation region, in water using solution NMR techniques. We provide structural information on 2'-OH hydroxyl groups in the form of orientational preferences contradicting the paradigm that the 2'-OH hydroxyl typically points away from the ribose H1' proton.     

Fast interstrand cross-linking of cisplatin-DNA monoadducts compared with intrastrand chelation: a kinetic study using hairpin-stabilized duplex oligonucleotides

The antitumor drug cisplatin forms two kinds of guanine-guanine cross-links with DNA: intrastrand, occurring mainly at GG sites, and interstrand, formed at GC sites. The former are generally more abundant than the latter, at least in experiments with linear duplex DNA. The formation of interstrand cross-links requires partial disruption of the Watson-Crick base pairing, and one could therefore expect the cross-linking reaction to be rather slow. In contrast with this expectation, kinetic measurements reported here indicate that interstrand cross-linking is as fast as intrastrand, or even faster. We have investigated the reactions between two hairpin-stabilized DNA duplexes, containing either a d(TGCA)(2) sequence (duplex TGCA) or a d(G(1)G(2)CA)-d(TG(3)CC) sequence (duplex GGCA), and the diaqua form of cisplatin, cis-[Pt(NH(3))(2)(H(2)O)(2)](2+), in an unbuffered solution kept at pH 4.5 +/- 0.1 and 20 degrees C. Using HPLC as the analytical method, we have determined the platination (first step) and chelation (second step) rate constants for these reaction systems. Duplex TGCA, in which the two guanines are quasi-equivalent, is found to be platinated very slowly (k=0.5 +/- 0.1M(-1)s(-1)) and to form the final interstrand cross-link very rapidly (k=13 +/- 3 x 10(-3) s(-11)). For GGCA, we find that G(1) is platinated rapidly (k=32 +/- 5M(-1)s(-1)) to form a long-lived monoadduct, which is only slowly chelated (k=0.039 +/- 0.001 x 10(-3) s(-1)) by G(2) (intrastrand), while G(2) is platinated one order of magnitude more slowly than G(1) (k=2.0 +/- 0.5M(-1)s(-1)) and chelated fairly rapidly both by G(1) (intrastrand: k=0.4 +/-0.1 x 10(-3) s(-1)) and G(3) (interstrand: k=0.2 +/- 0.1 x 10(-3) s(-1)); finally, G(3) is platinated at about the same rate as G(2) (k=2.4 +/- 0.5M(-1)s(-1)) and chelated very rapidly by G(2) (interstrand: k=10 +/- 4 x 10(-3) s(-1)). These results suggest that the low occurrence of interstrand cross-links in cisplatinated DNA is due to an extremely slow initial platination of guanines involved in d(GC)(2) sequences, rather than to a slow cross-linking reaction.     

Chemical shift tensors of protonated base carbons in helical RNA and DNA from NMR relaxation and liquid crystal measurements

Knowledge of (13)C chemical shift anisotropy (CSA) tensors in nucleotide bases is important for interpretation of NMR relaxation data in terms of local dynamic properties of nucleic acids and for analysis of residual chemical shift anisotropy (RCSA) resulting from weak alignment. CSA tensors for protonated nucleic acid base carbons have been derived from measurements on a uniformly (13)C-enriched helical A-form RNA segment and a helical B-form DNA dodecamer at natural (13)C abundance. The magnitudes of the derived CSA principal values are tightly restricted by the magnetic field dependencies of the (13)C transverse relaxation rates, whereas the tensor orientation and asymmetry follow from quantitative measurements of interference between (13)C-{(1)H} dipolar and (13)C CSA relaxation mechanisms. Changes in the chemical shift between the isotropic and aligned states, Deltadelta, complement these measurements and permit cross-validation. The CSA tensors are determined from the experimental Deltadelta values and relaxation rates, under the assumption that the CSA tensor of any specific carbon in a given type of base is independent of the base position in either the RNA or DNA helix. However, the experimental data indicate that for pyrimidine C(6) carbons in A-form RNA the CSA magnitude is considerably larger than in B-form DNA. This result is supported by quantum chemical calculations and is attributed in part to the close proximity between intranucleotide C(6)H and O(5)' atoms in RNA. The magnitudes of the measured CSA tensors, on average, agree better with previous solid-state NMR results obtained on powdered nucleosides than with prior results from quantum chemical calculations on isolated bases, which depend rather strongly on the level of theory at which the calculations are carried out. In contrast, previously computed orientations of the chemical shift tensors agree well with the present experimental results and exhibit less dependence on the level of theory at which the computations are performed.     

Long-range oxidative damage to DNA: effects of distance and sequence

INTRODUCTION: Oxidative damage to DNA in vivo can lead to mutations and cancer. DNA damage and repair studies have not yet revealed whether permanent oxidative lesions are generated by charges migrating over long distances. Both photoexcited *Rh(III) and ground-state Ru(III) intercalators were previously shown to oxidize guanine bases from a remote site in oligonucleotide duplexes by DNA-mediated electron transfer. Here we examine much longer charge-transport distances and explore the sensitivity of the reaction to intervening sequences. RESULTS: Oxidative damage was examined in a series of DNA duplexes containing a pendant intercalating photooxidant. These studies revealed a shallow dependence on distance and no dependence on the phasing orientation of the oxidant relative to the site of damage, 5'-GG-3'. The intervening DNA sequence has a significant effect on the yield of guanine oxidation, however. Oxidation through multiple 5'-TA-3' steps is substantially diminished compared to through other base steps. We observed intraduplex guanine oxidation by tethered *Rh(III) and Ru(III) over a distance of 200 A. The distribution of oxidized guanine varied as a function of temperature between 5 and 35 degrees C, with an increase in the proportion of long-range damage (> 100 A) occurring at higher temperatures. CONCLUSIONS: Guanines are oxidized as a result of DNA-mediated charge transport over significant distances (e.g. 200 A). Although long-range charge transfer is dependent on distance, it appears to be modulated by intervening sequence and sequence-dependent dynamics. These discoveries hold important implications with respect to DNA damage in vivo.     

Structural studies of LNA:RNA duplexes by NMR: conformations and implications for RNase H activity

We have used NMR and CD spectroscopy to study the conformations of modified oligonucleotides (locked nucleic acid, LNA) containing a conformationally restricted nucleotide (T(L)) with a 2'-O,4'-C-methylene bridge. We have investigated two LNA:RNA duplexes, d(CTGAT(L)ATGC):r(GCAUAUCAG) and d(CT(L)GAT(L)AT(L)GC):r(GCAUAUCAG), along with the unmodified DNA:RNA reference duplex. Increases in the melting temperatures of +9.6 degrees C and +8.1 degrees C per modification relative to the unmodified duplex were observed for these two LNA:RNA sequences. The three duplexes all adopt right-handed helix conformations and form normal Watson-Crick base pairs with all the bases in the anti conformation. Sugar conformations were determined from measurements of scalar coupling constants in the sugar rings and distance information derived from 1H-1H NOE measurements; all the sugars in the RNA strands of the three duplexes adopt an N-type conformation (A-type structure), whereas the sugars in the DNA strands change from an equilibrium between S- and N-type conformations in the unmodified duplex towards more of the N-type conformation when modified nucleotides are introduced. The presence of three modified T(L) nucleotides induces drastic conformational shifts of the remaining unmodified nucleotides of the DNA strand, changing all the sugar conformations except those of the terminal sugars to the N type. The CD spectra of the three duplexes confirm the structural changes described above. On the basis of the results reported herein, we suggest that the observed conformational changes can be used to tune LNA:RNA duplexes into substrates for RNase H: Partly modified LNA:RNA duplexes may adopt a duplex structure between the standard A and B types, thereby making the RNA strand amenable to RNase H-mediated degradation.     

Pathways of polaron and bipolaron transport in DNA double strands

We investigate the pathways of polaron and bipolaron transports in DNA double strands with an extended Su-Schrieffer-Heeger model involving the effects of solvent polarization. We find that the long-range transport of polaron/bipolaron under high electric field at low temperature is the field-facilitated sequential tunneling through spatial-disordered potential barriers via multiple intrastrand and interstrand pathways. Although the interstrand pathways may be very active and effective in some DNA sequences, the intrastrand ones always dominate the charge transfer when the excess charge moves close to the final acceptor.     

Molecular dynamics studies of ion distributions for DNA duplexes and DNA clusters: salt effects and connection to DNA melting

We present extensive molecular dynamics simulations of the ion distributions for DNA duplexes and DNA clusters using the Amber force field with implicit water. The distribution of ions and the electrostatic energy of ions around an isolated DNA duplex and clusters of DNA duplexes in different salt (NaCl) concentrations over the range 0.2-1.0 mol/L are determined on the basis of the simulation results. Using the electrostatic energy profile, we determine a local net charge fraction phi, which is found to increase with increasing of salt concentration. For DNA clusters containing two DNA duplexes (DNA pair) or four DNA duplexes, phi increases as the distance between the duplexes decreases. Combining this result with experimental results for the dependence of the DNA melting temperature on bulk salt concentration, we conclude that for a pair of DNA duplexes the melting temperature increases by 5-10 K for interaxis separations of 25-40 A. For a cluster of four DNA duplexes, an even larger melting temperature increase should occur. We argue that this melting temperature increase in dense DNA clusters is responsible for the cooperative melting mechanism in DNA-linked nanoparticle aggregates and DNA-linked polymer aggregates.     

How do size-expanded DNA nucleobases enhance duplex stability? Computational analysis of the hydrogen-bonding and stacking ability of xDNA bases

Computational chemistry (B3LYP, MP2) is used to study the properties of size-expanded DNA nucleobases generated by inserting a benzene spacer into the natural nucleobases. Although the addition of the spacer does not significantly affect the hydrogen-bonding properties of natural nucleobases, the orientation of the base about the glycosidic bond necessary for Watson-Crick binding is destabilized, which could have implications for the selectivity of expanded bases, as well as the stability of expanded duplexes. Consideration of the (stacked) binding energies in the preferred relative orientation of natural and expanded nucleobases aligned according to their centers of mass reveals that the stacking within natural dimers can be increased by up to 50% upon expansion of one nucleobase and up to 90% upon expansion of two nucleobases. The implications of these findings to the stability of expanded duplexes were revealed by considering simplified models of natural and mixed duplexes composed of four nucleobases. Although intra- and interstrand interactions within double helices are typically less than those predicted when nucleobases are stacked according to their centers of mass, some nucleobases utilize their full stacking potential within double helices, where both intra- and interstrand interactions can be significant. Most importantly, increasing the size of nucleobases within the duplex significantly increases both intra- and interstrand stacking interactions. Specifically, some interactions are double the magnitude of the corresponding intrastrand interactions in natural helices, and even greater increases in interstrand interactions are sometimes found. Thus, our work suggests that mixed duplexes composed of natural bases hydrogen bound to expanded bases may exploit the increase in the inherent stacking ability of the expanded bases in more than one way and thereby afford duplexes with greater stability than natural DNA.     

Unique properties of DNA interstrand cross-links of antitumor oxaliplatin and the effect of chirality of the carrier ligand

The different antitumor and other biological effects of the third-generation antitumor platinum drug oxaliplatin [(1R,2R-diamminocyclohexane)oxalatoplatinum(II)] in comparison with those of conventional cisplatin [cis-diamminedichloridoplatinum(II)] are often explained by the ability of oxaliplatin to form DNA adducts of different conformation and consequently to exhibit different cytotoxic effects. This work describes, for the first time, the structural and biochemical characteristics of the interstrand cross-links of oxaliplatin. We find that: 1) DNA bending, unwinding, thermal destabilization, and delocalization of the conformational alteration induced by the cross-link of oxaliplatin are greater than those observed with the cross-link of cisplatin; 2) the affinity of high-mobility-group proteins (which are known to mediate the antitumor activity of platinum complexes) for the interstrand cross-links of oxaliplatin is markedly lower than for those of cisplatin; and 3) the chirality at the carrier 1,2-diaminocyclohexane ligand can affect some important structural properties of the interstrand cross-links of cisplatin analogues. Thus, the information contained in the present work is also useful for a better understanding of how the stereochemistry of the carrier amine ligands of cisplatin analogues can modulate their anticancer and mutagenic properties. The significance of this study is also reinforced by the fact that, in general, interstrand cross-links formed by various compounds of biological significance result in greater cytotoxicity than is expected for monofunctional adducts or other intrastrand DNA lesions. Therefore, we suggest that the unique properties of the interstrand cross-links of oxaliplatin are at least partly responsible for this drug's unique antitumor effects.