Combined theoretical and computational study of interstrand DNA guanine-guanine cross-linking by trans-[Pt(pyridine)2] derived from the photoactivated prodrug trans,trans,trans-[Pt(N3)2(OH)2(pyridine)2

Molecular modeling and extensive experimental studies are used to study DNA distortions induced by binding platinum(II)-containing fragments derived from cisplatin and a new class of photoactive platinum anticancer drugs. The major photoproduct of the novel platinum(IV) prodrug trans,trans,trans-[Pt(N(3))(2)(OH)(2)(py)(2)] (1) contains the trans-{Pt(py)(2)}(2+) moiety. Using a tailored DNA sequence, experimental studies establish the possibility of interstrand binding of trans-{Pt(py)(2)}(2+) (P) to guanine N7 positions on each DNA strand. Ligand field molecular mechanics (LFMM) parameters for Pt-guanine interactions are then derived and validated against a range of experimental structures from the Cambridge Structural Database, published quantum mechanics (QM)/molecular mechanics (MM) structures of model Pt-DNA systems and additional density-functional theory (DFT) studies. Ligand field molecular dynamics (LFMD) simulation protocols are developed and validated using experimentally characterized bifunctional DNA adducts involving both an intra- and an interstrand cross-link of cisplatin. We then turn to the interaction of P with the DNA duplex dodecamer, d(5'-C(1)C(2)T(3)C(4)T(5)C(6)G(7)T(8)C(9)T(10)C(11)C(12)-3')·d(5'-G(13)G(14)A(15)G(16)A(17)C(18)G(19)A(20)G(21)A(22)G(23)G(24)-3') which is known to form a monofunctional adduct with cis-{Pt(NH(3))(2)(py)}. P coordinated to G(7) and G(19) is simulated giving a predicted bend toward the minor groove. This is widened at one end of the platinated site and deepened at the opposite end, while the P-DNA complex exhibits a global bend of ～67° and an unwinding of ～20°. Such cross-links offer possibilities for specific protein-DNA interactions and suggest possible mechanisms to explain the high potency of this photoactivated complex.     

Selective G-quadruplex DNA stabilizing agents based on bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine

Various biologically relevant G-quadruplex DNA structures offer a platform for therapeutic intervention for altering the gene expression or by halting the function of proteins associated with telomeres. One of the prominent strategies to explore the therapeutic potential of quadruplex DNA structures is by stabilizing them with small molecule ligands. Here we report the synthesis of bisquinolinium and bispyridinium derivatives of 1,8-naphthyridine and their interaction with human telomeric DNA and promoter G-quadruplex forming DNAs. The interactions of ligands with quadruplex forming DNAs were studied by various biophysical, biochemical, and computational methods. Results indicated that bisquinolinium ligands bind tightly and selectively to quadruplex DNAs at low ligand concentration (～0.2-0.4 μM). Furthermore, thermal melting studies revealed that ligands imparted higher stabilization for quadruplex DNA (an increase in the T(m) of up to 21 °C for human telomeric G-quadruplex DNA and >25 °C for promoter G-quadruplex DNAs) than duplex DNA (ΔT(m) ≤ 1.6 °C). Molecular dynamics simulations revealed that the end-stacking binding mode was favored for ligands with low binding free energy. Taken together, the results indicate that the naphthyridine-based ligands with quinolinium and pyridinium side chains form a promising class of quadruplex DNA stabilizing agents having high selectivity for quadruplex DNA structures over duplex DNA structures.     

Interconversion of the cis-5R,6S- and trans-5R,6R-thymine glycol lesions in duplex DNA

Thymine glycol (Tg), 5,6-dihydroxy-5,6-dihydrothymine, is formed in DNA by the reaction of thymine with reactive oxygen species. The 5R Tg lesion was incorporated site-specifically into 5'-d(G(1)T(2)G(3)C(4)G(5)Tg(6)G(7)T(8)T(9)T(10)G(11)T(12))-3'; Tg = 5R Tg. The Tg-modified oligodeoxynucleotide was annealed with either 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)A(19)C(20)G(21)C(22)A(23)C(24))-3', forming the Tg(6) x A(19) base pair, corresponding to the oxidative damage of thymine in DNA, or 5'-d(A(13)C(14)A(15)A(16)A(17)C(18)G(19)C(20)G(21)C(22)A(23)C(24))-3', forming the mismatched Tg(6) x G(19) base pair, corresponding to the formation of Tg following oxidative damage and deamination of 5-methylcytosine in DNA. At 30 degrees C, the equilibrium ratio of cis-5R,6S:trans-5R,6R epimers was 7:3 for the duplex containing the Tg(6) x A (19) base pair. In contrast, for the duplex containing the Tg(6) x G(19) base pair, the cis-5R,6S:trans-5R,6R equilibrium favored the cis-5R,6S epimer; the level of the trans-5R,6R epimer remained below the level of detection by NMR. The data suggested that Tg disrupted hydrogen bonding interactions, either when placed opposite to A(19) or G(19). Thermodynamic measurements indicated a 13 degrees C reduction of T(m) regardless of whether Tg was placed opposite dG or dA in the complementary strand. Although both pairings increased the free energy of melting by 3 kcal/mol, the melting of the Tg x G pair was more enthalpically favored than was the melting of the Tg x A pair. The observation that the position of the equilibrium between the cis-5R,6S and trans-5R,6R thymine glycol epimers in duplex DNA was affected by the identity of the complementary base extends upon observations that this equilibrium modulates the base excision repair of Tg [Ocampo-Hafalla, M. T.; Altamirano, A.; Basu, A. K.; Chan, M. K.; Ocampo, J. E.; Cummings, A., Jr.; Boorstein, R. J.; Cunningham, R. P.; Teebor, G. W. DNA Repair (Amst) 2006, 5, 444-454].     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.     

Study of binding affinity and selectivity of perylene and coronene derivatives towards duplex and quadruplex DNA by ESI‐MS

In this paper, we report an extensive electrospray ionization mass spectrometry (ESI‐MS) study of the noncovalent interactions between different intermolecular and intramolecular G‐quadruplex structures and several perylene and coronene ligands. The selectivity of these compounds toward quadruplex structures with respect to duplex DNA, a fundamental topic for the biological evaluation and the pharmacological application of these ligands as potential chemotherapeutic agents, has also been investigated. After exploring this topic according to the classical approach based on the very simple duplex model of an autocomplementary dodecamer, we extended our analysis reporting for the first time a competition ESI‐MS experiment in the presence of genomic DNA fragments. Whereas those ligands showing a high level of selectivity between quadruplex and duplex oligonucleotides, in terms of binding constants and percentage of bound DNA, confirmed their selectivity in the competition experiment, the contrary was not always true: some ligands showing poor selectivity with the autocomplementary dodecamer resulted selective in the presence of genomic DNA fragments. This result suggests that physiologically nonrelevant interactions are possible with a short duplex oligonucleotide. This means that the dodecamer can fail in representing a biologically significant structural model, or, better, that it can be used to quickly screen potentially selective molecules, but bearing in mind the high probability of false negative results. Copyright © 2008 John Wiley & Sons, Ltd.     

Stabilizing or destabilizing oligodeoxynucleotide duplexes containing single 2'-deoxyuridine residues with 5-alkynyl substituents

The 5-position of pyrimidines in DNA duplexes offers a site for introducing alkynyl substituents that protrude into the major groove and thus do not sterically interfere with helix formation. Substituents introduced at the 5-position of the deoxyuridine residue of dU:dA base pairs may stabilize duplexes and reinforce helices weakened by a low G/C content, which would otherwise lead to false negative results in DNA chip experiments. Here we report on a method for preparing oligonucleotides with a 5-alkynyl substituent at a 2'-deoxyuridine residue by on-support Sonogashira coupling involving the fully assembled oligonucleotide. A total of 25 oligonucleotides with 5-alkynyl substituents were prepared. The substituents either decrease the UV melting point of the duplex with the complementary strand or increase it by up to 7.1 degrees C, compared with that of the unmodified control duplex. The most duplex-stabilizing substituent, a pyrenylbutyramidopropyne moiety, is likely to intercalate but does not prevent sequence-specific base pairing of the modified deoxyuridine residue or the neighboring nucleotides. It also increases the signal for a target strand when employed on a small oligonucleotide microarray. The ability to tune the melting point of a DNA dodecamer duplex with a single side chain over a temperature range of >11 degrees C may prove useful when developing DNA sequences for biomedical applications.     

Sequence-dependent bending of DNA induced by cisplatin: NMR structures of an A.T-rich 14-mer duplex

The NMR solution structure of the A.T rich DNA 14-mer duplex d(ATACATGGTACATA).d(TATGTACCATGTAT) is reported. This is compared with the NMR structure of the same duplex intrastrand cross-linked at the d(G*pG*) site by cis-(Pt(NH3)2?2+, derived from the anticancer drug cisplatin. The unmodified duplex has B-DNA geometry, but there is a large positive base-pair roll (roll angle 24 +/- 2 degrees) at the T9-A10 step on the 3' side of the central GG site. Platination of the DNA duplex causes the adjacent guanine bases to roll toward one another (roll angle 44 +/- 4 degrees), leading to an overall helix bend of 52 +/- 9 degrees. The platinum atom is displaced from the planes of the coordinated G7* and G8* by 0.8 A and 0.3 A, respectively. The minor groove opposite the platinum lesion is widened and flattened, with geometric parameters similar to those of A-form DNA. The unwinding of the helix at the platination site is 26 degrees. Platination causes the DNA duplex to bend toward the 3'-end (with respect to the G*G* strand), in contrast to G C-rich structures reported previously, which bend toward the 5'-end. This difference can be attributed to the predisposition of the A.T rich duplex toward bending in this region. Protein recognition of bent platinated G*G* lesions may therefore exhibit a strong dependence on the local DNA structure.     

Marked dependence on carrier-ligand bulk but not on carrier-ligand chirality of the duplex versus single-strand forms of a DNA oligonucleotide with a series of G-Pt(II)-G intrastrand cross-links modeling cisplatin-DNA adducts

The N7-Pt-N7 adjacent G,G intrastrand DNA cross-link responsible for cisplatin anticancer activity is dynamic, promotes local "melting" in long DNA, and converts many oligomer duplexes to single strands. For 5'-d(A1T2G3G4G5T6A7C8C9C10A11T12)-3' (G3), treatment of the (G3)2 duplex with five pairs of [LPt(H2O)2]2+ enantiomers (L = an asymmetric diamine) formed mixtures of LPt-G3 products (1 Pt per strand) cross-linked at G3,G4 or at G4,G5 in all cases. L chirality exerted little influence. For primary diamines L with bulk on chelate ring carbons (e.g., 1,2-diaminocyclohexane), the duplex was converted completely into single strands (G3,G4 coils and G4,G5 hairpins), exactly mirroring results for cisplatin, which lacks bulk. In sharp contrast, for secondary diamines L with bulk on chelate ring nitrogens (e.g., 2,2'-bipiperidine, Bip), unexpectedly stable duplexes having two platinated strands (even a unique G3,G4/G4,G5 heteroduplex) were formed. After enzymatic digestion of BipPt-G3 duplexes, the conformation of the relatively nondynamic G,G units was shown to be head-to-head (HH) by HPLC/mass spectrometric characterization. Because the HH conformation dominates at the G,G lesion in duplex DNA and in the BipPt-G3 duplexes, the stabilization of the duplex form only when the L nitrogen adducts possess bulk suggests that H-bonding interactions of the Pt-NH groups with the flanking DNA lead to local melting and to destabilization of oligomer duplexes. The marked dependence of adduct properties on L bulk and the minimal dependence on L chirality underscore the need for future exploration of the roles of the L periphery in affecting anticancer activity.     

Quadruplex formation by a guanine-rich PNA oligomer

A guanine-rich PNA dodecamer having the sequence H-G4T4G4-Lys-NH2 (G-PNA) hybridizes with a DNA dodecamer of homologous sequence to form a four-stranded quadruplex (Datta, B.; Schmitt, C.; Armitage, B. A. J. Am. Chem. Soc. 2003, 125, 4111-4118). This report describes quadruplex formation by the PNA alone. UV melting curves and fluorescence resonance energy transfer experiments reveal formation of a multistranded structure stabilized by guanine tetrads. The ion dependency of these structures is analogous to that reported for DNA quadruplexes. Electrospray ionization mass spectrometry indicates that both dimeric and tetrameric quadruplexes are formed by G4-PNA, with the dimeric form being preferred. These results have implications for the use of G-rich PNA for homologous hybridization to G-rich targets in chromosomal DNA and suggest additional applications in assembling quadruplex structures within lipid bilayer environments.     

Spectroscopic characterization of interstrand carbinolamine cross-links formed in the 5'-CpG-3' sequence by the acrolein-derived gamma-OH-1,N2-propano-2'-deoxyguanosine DNA adduct

The interstrand N2,N2-dG DNA cross-linking chemistry of the acrolein-derived gamma-OH-1,N2-propanodeoxyguanosine (gamma-OH-PdG) adduct in the 5'-CpG-3' sequence was monitored within a dodecamer duplex by NMR spectroscopy, in situ, using a series of site-specific 13C- and 15N-edited experiments. At equilibrium 40% of the DNA was cross-linked, with the carbinolamine form of the cross-link predominating. The cross-link existed in equilibrium with the non-crosslinked N2-(3-oxo-propyl)-dG aldehyde and its geminal diol hydrate. The ratio of aldehyde/diol increased at higher temperatures. The 1,N2-dG cyclic adduct was not detected. Molecular modeling suggested that the carbinolamine linkage should be capable of maintaining Watson-Crick hydrogen bonding at both of the tandem C x G base pairs. In contrast, dehydration of the carbinolamine cross-link to an imine (Schiff base) cross-link, or cyclization of the latter to form a pyrimidopurinone cross-link, was predicted to require disruption of Watson-Crick hydrogen bonding at one or both of the tandem cross-linked C x G base pairs. When the gamma-OH-PdG adduct contained within the 5'-CpG-3' sequence was instead annealed into duplex DNA opposite T, a mixture of the 1,N2-dG cyclic adduct, the aldehyde, and the diol, but no cross-link, was observed. With this mismatched duplex, reaction with the tetrapeptide KWKK formed DNA-peptide cross-links efficiently. When annealed opposite dA, gamma-OH-PdG remained as the 1,N2-dG cyclic adduct although transient epimerization was detected by trapping with the peptide KWKK. The results provide a rationale for the stability of interstrand cross-links formed by acrolein and perhaps other alpha,beta-unsaturated aldehydes. These sequence-specific carbinolamine cross-links are anticipated to interfere with DNA replication and contribute to acrolein-mediated genotoxicity.     

The role of organic linkers in directing DNA self-assembly and significantly stabilizing DNA duplexes

We show a simple method to control both the stability and the self-assembly behavior of DNA structures. By connecting two adjacent duplexes with small synthetic linkers, factors such as linker rigidity and DNA strand orientation can increase the thermal denaturation temperature of 17 base-pair duplexes by up to 10 °C, and significantly increase the cooperativity of melting of the two duplexes. The same DNA sequence can thus be tuned to melt at vastly different temperatures by selecting the linker structure and DNA-to-linker connectivity. In addition, a small rigid m-triphenylene linker directly affects the self-assembly product distribution. With this linker, changes in the orientation of the linked strands (e.g., 5'3' vs 3'3') can lead to dramatic changes in the self-assembly behavior, from the formation of cyclic dimer and tetramer to higher-order oligomers. These variations can be readily predicted using a simple strand-end alignment model.     

Electrochemiluminescent monomers for solid support syntheses of Ru(II)-PNA bioconjugates: multimodal biosensing tools with enhanced duplex stability

The feasibility of devising a solid support mediated approach to multimodal Ru(II)-peptide nucleic acid (PNA) oligomers is explored. Three Ru(II)-PNA-like monomers, [Ru(bpy)(2)(Cpp-L-PNA-OH)](2+) (M1), [Ru(phen)(2)(Cpp-L-PNA-OH)](2+) (M2), and [Ru(dppz)(2)(Cpp-L-PNA-OH)](2+) (M3) (bpy = 2,2'-bipyridine, phen = 1,10-phenanthroline, dppz = dipyrido[3,2-a:2',3'-c]phenazine, Cpp-L-PNA-OH = [2-(N-9-fluorenylmethoxycarbonyl)aminoethyl]-N-[6-(2-(pyridin-2yl)pyrimidine-4-carboxamido)hexanoyl]-glycine), have been synthesized as building blocks for Ru(II)-PNA oligomers and characterized by IR and (1)H NMR spectroscopy, mass spectrometry, electrochemistry and elemental analysis. As a proof of principle, M1 was incorporated on the solid phase within the PNA sequences H-g-c-a-a-t-a-a-a-a-Lys-NH(2) (PNA1) and H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-lys-NH(2) (PNA4) to give PNA2 (H-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)) and PNA3 (H-P-K-K-K-R-K-V-g-c-a-a-t-a-a-a-a-M1-lys-NH(2)), respectively. The two Ru(II)-PNA oligomers, PNA2 and PNA3, displayed a metal to ligand charge transfer (MLCT) transition band centered around 445 nm and an emission maximum at about 680 nm following 450 nm excitation in aqueous solutions (10 mM PBS, pH 7.4). The absorption and emission response of the duplexes formed with the cDNA strand (DNA: 5'-T-T-T-T-T-T-T-A-T-T-G-C-T-T-T-3') showed no major variations, suggesting that the electronic properties of the Ru(II) complexes are largely unaffected by hybridization. The thermal stability of the PNA·DNA duplexes, as evaluated from UV melting experiments, is enhanced compared to the corresponding nonmetalated duplexes. The melting temperature (T(m)) was almost 8 °C higher for PNA2·DNA duplex, and 4 °C for PNA3·DNA duplex, with the stabilization attributed to the electrostatic interaction between the cationic residues (Ru(II) unit and positively charged lysine/arginine) and the polyanionic DNA backbone. In presence of tripropylamine (TPA) as co-reactant, PNA2, PNA3, PNA2·DNA and PNA3·DNA displayed strong electrochemiluminescence (ECL) signals even at submicromolar concentrations. Importantly, the combination of spectrochemical, thermal and ECL properties possessed by the Ru(II)-PNA sequences offer an elegant approach for the design of highly sensitive multimodal biosensing tools.     

Gold nanoparticles-based colorimetric investigation of triplex formation under weak alkalic pH environment with the aid of Ag+

A novel colorimetric method for investigating triplex formation between oligonucleotide modified Au nanoparticles (AuNPs) under weak alkalic pH environment is developed based upon the specific recognition property of Ag+ with CGC triads. Oligonucleotide 5'-SH-T12-CTTCTTTCCTTTCTTC-3' (oligo-1) is modified on the surface of AuNPs. Upon addition of oligonucleotide 5'-GAAGAAAGGAAAGAAG-3' (oligo-2), triplex formation between oligo-1 modified AuNPs occurred at pH 8.0 with the aid of Ag+, triggers the aggregation of AuNPs, accompany with the solution color change from red to purple. The melting temperature demonstrates a 31 °C increase for the triplex DNA compose of 10 T?A°T triads and 6 C?G°C triads upon addition of Ag+, the disassociation constant (Kd) between Ag+ and C?G°C triads is 3.6 μM. Moreover, triplex formation between AuNPs depending on Ag+ can be used to recognize Ag+ ion with the naked eye, as well as UV-vis absorption spectroscopy.     

Nucleic acid secondary structures containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine

Oligodeoxynucleotides containing the double-headed nucleoside 5'(S)-C-(2-(thymin-1-yl)ethyl)thymidine were prepared by standard solid phase synthesis. The synthetic building block for incorporating the double-headed moiety was prepared from thymidine, which was stereoselectively converted to a protected 5'(S)-C-hydroxyethyl derivative and used to alkylate the additional thymine by a Mitsunobu reaction. The oligodeoxynucleotides were studied in different nucleic acid secondary structures: duplexes, bulged duplexes, three-way junctions and artificial DNA zipper motifs. The thermal stability of these complexes was studied, demonstrating an almost uniform thermal penalty of incorporating one double-headed nucleoside moiety into a duplex or a bulged duplex, comparable to the effects of the previously reported double-headed nucleoside 5'(S)-C-(thymin-1-yl)methylthymidine. The additional base showed only very small effects when incorporated into DNA or RNA three-way junctions. The various DNA zipper arrangements indicated that extending the linker from methylene to ethylene almost completely removed the selective minor groove base-base stacking interactions observed for the methylene linker in a (-3)-zipper, whereas interactions, although somewhat smaller, were observed for the ethylene linker in a (-4)-zipper motif.     

A pyrazolato-bridged dinuclear platinum(II) complex induces only minor distortions upon DNA-binding

The cytotoxic, pyrazolato-bridged dinuclear platinum(II) complex [(cis-{Pt(NH3)2})2(mu-OH)(mu-pz)]2+ (pz=pyrazolate) has been found to cross-link two adjacent guanines of a double-stranded DNA decamer without destabilizing the duplex and without changing the directionality of the helix axis. A 1H NMR study of the oligonucleotide d(CTCTG*G*TCTC)-d(GAGACCAGAG), cross-linked at the two G* guanines by [(cis-{Pt(NH3)2})2(mu-pz)]3+, and molecular dynamics simulations of the explicitly solvated duplex were performed to characterize the structural details of the adduct. The dinuclear platinum cross-link unwinds the helix by approximately 15 degrees , that is, to a similar extent as the widely used antitumor drug cisplatin, but, in contrast to the latter, induces no significant bend in the helix axis. The Watson-Crick base-pairing remains intact, and the melting temperature of the duplex is unaffected by the cross-link. The helical twist is considerably reduced between the two platinated bases, as becomes manifest in an unusually short sequential H1'-H1' distance. This unwinding also affects the sugar ring of the guanosine in the 3'-position to the cross-link, which presents an N<-->S equilibrium. This is the first cytotoxic platinum complex that has been successfully designed by envisioning the structural consequences of its binding to DNA.     

Nature of minor-groove binders-DNA complexes in the gas phase

The structure of noncovalent complexes of DNA duplex with minor groove binders (mG-binders) has been analyzed by state of the art molecular dynamics (MD) simulations. More than 3.3 micros of MD trajectories (including 4 x 0.5 micros trajectories) were collected for the Dickerson's dodecamer bound to DAPI, Hoechst 33258, and Netropsin. Comparison of these trajectories with control simulations in water allowed us to determine that the extreme dehydration and partial neutralization occurring during electrospray experiments does not produce the disruption of the DNA:mG-binder complexes or the dissociation of the two strands of the duplex. Irrespective of the drug and the simulation conditions the mG-binders remains bound to the DNA near the preferential binding position in aqueous conditions. Large distortions appear in the two DNA strands, which maintain however a memory of the original DNA duplex structure in water, and a general helical-like conformation.     

Synthesis and pairing properties of oligodeoxynucleotides containing bicyclo-RNA and bicyclo-ANA modifications

The synthesis of the ribo(bc-rT)- and arabino(bc-araT)-version of bicyclothymidine (bc-dT) has been achieved. A conformational analysis by X-ray and/or (1)H NMR spectroscopy on the corresponding 3',5'-benzyl-protected nucleosides featured a rigid C(2')-endo conformation for the furanose ring, irrespective of the configuration of the OH group at C(2'). The conformation of the carbocyclic ring in these nucleosides was found to be less defined and thus more flexible. Both nucleosides were converted into the corresponding phosphoramidites and incorporated into oligodeoxynucleotides by standard DNA chemistry. T(m)-data of duplexes with cDNA and RNA revealed that a bc-rT unit strongly destabilized duplexes with cDNA and RNA by 6-8 °C/mod, while bc-araT was almost T(m) neutral. A rationale based on a previous structure of a bc-DNA mini duplex suggests that the strong destabilization caused by a bc-rT unit arises from unfavorable steric interactions of the equatorial 2'-OH group with the sugar residue of the 3'-neighboring nucleotide unit.     

Synthesis, gene silencing, and molecular modeling studies of 4'-C-aminomethyl-2'-O-methyl modified small interfering RNAs

The linear syntheses of 4'-C-aminomethyl-2'-O-methyl uridine and cytidine nucleoside phosphoramidites were achieved using glucose as the starting material. The modified RNA building blocks were incorporated into small interfering RNAs (siRNAs) by employing solid phase RNA synthesis. Thermal melting studies showed that the modified siRNA duplexes exhibited slightly lower T(m) (～1 °C/modification) compared to the unmodified duplex. Molecular dynamics simulations revealed that the 4'-C-aminomethyl-2'-O-methyl modified nucleotides adopt South-type conformation in a siRNA duplex, thereby altering the stacking and hydrogen-bonding interactions. These modified siRNAs were also evaluated for their gene silencing efficiency in HeLa cells using a luciferase-based reporter assay. The results indicate that the modifications are well tolerated in various positions of the passenger strand and at the 3' end of the guide strand but are less tolerated in the seed region of the guide strand. The modified siRNAs exhibited prolonged stability in human serum compared to unmodified siRNA. This work has implications for the use of 4'-C-aminomethyl-2'-O-methyl modified nucleotides to overcome some of the challenges associated with the therapeutic utilities of siRNAs.     

Neomycin-neomycin dimer: an all-carbohydrate scaffold with high affinity for AT-rich DNA duplexes

A dimeric neomycin-neomycin conjugate 3 with a flexible linker, 2,2'-(ethylenedioxy)bis(ethylamine), has been synthesized and characterized. Dimer 3 can selectively bind to AT-rich DNA duplexes with high affinity. Biophysical studies have been performed between 3 and different nucleic acids with varying base composition and conformation by using ITC (isothermal calorimetry), CD (circular dichroism), FID (fluorescent intercalator displacement), and UV (ultraviolet) thermal denaturation experiments. A few conclusions can be drawn from this study: (1) FID assay with 3 and polynucleotides demonstrates the preference of 3 toward AT-rich sequences over GC-rich sequences. (2) FID assay and UV thermal denaturation experiments show that 3 has a higher affinity for the poly(dA)·poly(dT) DNA duplex than for the poly(dA)·2poly(dT) DNA triplex. Contrary to neomycin, 3 destabilizes poly(dA)·2poly(dT) triplex but stabilizes poly(dA)·poly(dT) duplex, suggesting the major groove as the binding site. (3) UV thermal denaturation studies and ITC experiments show that 3 stabilizes continuous AT-tract DNA better than DNA duplexes with alternating AT bases. (4) CD and FID titration studies show a DNA binding site size of 10-12 base pairs/drug, depending upon the structure/sequence of the duplex for AT-rich DNA duplexes. (5) FID and ITC titration between 3 and an intramolecular DNA duplex [d(5'-A(12)-x-T(12)-3'), x = hexaethylene glycol linker] results in a binding stoichiometry of 1:1 with a binding constant ～10(8) M(-1) at 100 mM KCl. (6) FID assay using 3 and 512 hairpin DNA sequences that vary in their AT base content and placement also show a higher binding selectivity of 3 toward continuous AT-rich than toward DNA duplexes with alternate AT base pairs. (7) Salt-dependent studies indicate the formation of three ion pairs during binding of the DNA duplex d[5'-A(12)-x-T(12)-3'] and 3. (8) ITC-derived binding constants between 3 and DNA duplexes have the following order: AT continuous, d[5'-G(3)A(5)T(5)C(3)-3'] > AT alternate, d[5'-G(3)(AT)(5)C(3)-3'] > GC-rich d[5'-A(3)G(5)C(5)T(3)-3']. (9) 3 binds to the AT-tract-containing DNA duplex (B* DNA, d[5'-G(3)A(5)T(5)C(3)-3']) with 1 order of magnitude higher affinity than to a DNA duplex with alternating AT base pairs (B DNA, d[5'-G(3)(AT)(5)C(3)-3']) and with almost 3 orders of magnitude higher affinity than a GC-rich DNA (A-form, d[5'-A(3)G(5)C(5)T(3)-3']).     

Duplex stability of DNA.DNA and DNA.RNA duplexes containing 3'-S-phosphorothiolate linkages

3'-S-Phosphorothiolate (3'-SP) linkages have been incorporated into the DNA strand of both a DNA.RNA duplex and a DNA.DNA duplex. Thermal melting (T(m)) studies established that this modification significantly stabilises the DNA.RNA duplex with an average increase in T(m) of about 1.4 degrees C per modification. For two or three modifications, the increase in T(m) was larger for an alternating, as compared to the contiguous, arrangement. For more than three modifications their arrangement had no effect on T(m). In contrast to the DNA.RNA duplex, the 3'-S-phosphorothiolate linkage destabilised the DNA.DNA duplex, irrespective of the arrangement of the 3'-SP linkages. The effect of ionic strength on duplex stability was similar for both the phosphorothiolate-substituted and the unmodified RNA.DNA duplexes. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and comparisons are also drawn between the current study and those previously conducted with other modifications that have a similar conformational effect.