Sequence dependence of Pd(II)-mediated base pairing by palladacyclic nucleobase surrogates.

A C-nucleoside derivative of phenylpyridine or the respective palladacycle was incorporated at either 3'- or 5'-terminus of a short oligodeoxynucleotide. Hybridization properties of these modified oligonucleotides were studied in a fluorescence-based competition assay in addition to conventional UV melting temperature analysis and compared with those of a previously prepared analogue featuring the modified nucleoside in the middle of the sequence. With the unpalladated phenylpyridine oligonucleotides, UV melting temperature qualitatively correlated with the ability to displace a strand from a double helix in the competition assay, decreasing in the order 5' > 3' > middle. Corresponding results on the palladacyclic oligonucleotides were more difficult to interpret but both UV melting and competition experiments revealed a decrease in the duplex stability upon palladation in most cases. On the other hand, dependence of the UV melting temperature on the identity of the canonical nucleobase opposite to the modified nucleobase analogue was much more pronounced with the palladacyclic duplexes than with their unpalladated counterparts. Furthermore, UV melting profiles of the palladacyclic duplexes featured an additional transition at a temperature exceeding the melting temperature of the unmodified part of the duplex. Taken together, these results lend support to the idea of Pd(II)-mediated base pairs that are highly stable but incompatible with the geometry of a double helix.

2-Amino-1,3-benzothiazole-6-carboxamide Preferentially Binds the Tandem Mismatch Motif r(UY:GA).

RNA helices are often punctuated with non-Watson-Crick features that may be targeted by chemical compounds, but progress toward identifying such compounds has been slow. We embedded a tandem UU:GA mismatch motif (5'-UG-3':5'-AU-3') within an RNA hairpin stem to identify compounds that bind the motif specifically. The three-dimensional structure of the RNA hairpin and its interaction with a small molecule identified through virtual screening are presented. The G-A mismatch forms a sheared pair upon which the U-U base pair stacks. The hydrogen bond configuration of the U-U pair involves O2 of the U adjacent to the G and O4 of the U adjacent to the A. The G-A and U-U pairs are flanked by A-U and G-C base pairs, respectively, and the stability of the mismatch is greater than when the motif is within the context of other flanking base pairs or when the 5'-3' orientation of the G-A and U-U pairs is swapped. Residual dipolar coupling constants were used to generate an ensemble of structures against which a virtual screen of 64480 small molecules was performed. The tandem mismatch was found to be specific for one compound, 2-amino-1,3-benzothiazole-6-carboxamide, which binds with moderate affinity but extends the motif to include the flanking A-U and G-C base pairs. The finding that the affinity for the UU:GA mismatch is dependent on flanking sequence emphasizes the importance of the motif context and potentially increases the number of small noncanonical features within RNA that can be specifically targeted by small molecules.

Intercalation of a flavonoid, silibinin into DNA base pairs: Experimental and theoretical approach.

Polyphenols are secondary plant metabolites, which have received much attention because of their potential health benefits. Silibinin (SIL) is a well-known naturally occurring flavonolignan, which is extensively used in treating a wide variety of diseases as a dietary supplement as well as a prescribed drug. The mechanism of binding of SIL to calf thymus DNA (ctDNA) was investigated by employing multispectroscopic techniques, viz., absorption, fluorescence, and circular dichroism besides viscosity measurements and docking studies. Analysis of fluorescence results indicated that SIL has interacted with ctDNA and quenched its intensity through static quenching mechanism. The binding constant at room temperature was found to be 2.48×104 mol-1 , suggesting moderate binding affinity between SIL and ctDNA. The hypochromicity observed in the absorption spectra of ctDNA in the presence of SIL revealed the intercalation of SIL into ctDNA base pairs. Further, the intercalative mode of binding between SIL and ctDNA was confirmed by viscosity measurements and molecular docking studies. The outcome of present study helps to decipher the interaction mechanism between SIL and DNA at physiological pH, which further assists in the design of a new analogue for better therapeutic effects.

A DNA G-quadruplex/i-motif hybrid.

DNA can form many structures beyond the canonical Watson-Crick double helix. It is now clear that noncanonical structures are present in genomic DNA and have biological functions. G-rich G-quadruplexes and C-rich i-motifs are the most well-characterized noncanonical DNA motifs that have been detected in vivo with either proscribed or postulated biological roles. Because of their independent sequence requirements, these structures have largely been considered distinct types of quadruplexes. Here, we describe the crystal structure of the DNA oligonucleotide, d(CCAGGCTGCAA), that self-associates to form a quadruplex structure containing two central antiparallel G-tetrads and six i-motif C-C+ base pairs. Solution studies suggest a robust structural motif capable of assembling as a tetramer of individual strands or as a dimer when composed of tandem repeats. This hybrid structure highlights the growing structural diversity of DNA and suggests that biological systems may harbor many functionally important non-duplex structures.

Monitoring DNA-Ligand Interactions in Living Human Cells Using NMR Spectroscopy.

Studies on DNA-ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA-ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA-ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

Unraveling the Modulation of the Activity in Drugs Based on Methylated Phenanthroline When Intercalating between DNA Base Pairs.

Phenanthroline derivatives intercalate between base pairs of DNA and produce cytotoxic effects against tumoral cells. Nevertheless, modulation of their efficiency by substitution remains unclear in bibliography. In this work, the effects of methylation of phenanthroline, in number and position, when it intercalates between guanine-cytosine base pairs (GC/CG), were studied with PM6-DH2 and DFT-D methods including dispersion corrections. An analysis of the geometries, electronic structure, and energetics in the interaction was carried out for the studied systems. Our results were compared to experimental works to gain insight on the relation structure-interaction for the intercalated system with cytotoxicity. The trends are explained including not only intrinsic contributions to energy, ΔEPauli, ΔEdisp, ΔEorb, and ΔEelstat, but also the solvation energy, ΔESolv. A subtle balance between the number of stabilizing weak interactions (CH/π, CH/n, etc.) and steric hindrance seems to be related to the efficiency of such drugs.

3-Fluoro-2-mercuri-6-methylaniline Nucleotide as a High-Affinity Nucleobase-Specific Hybridization Probe.

A 3-fluoro-6-methylaniline nucleoside was synthesized and incorporated into an oligonucleotide, and its ability to form mercury-mediated base pairs was studied. UV melting experiments revealed increased duplex stability with thymine, guanine, and cytosine opposite to the probe and a clear nucleobase-specific binding preference (T > G > C > A). Moreover, the 3-fluoro group was utilized as a spin label that showed distinct 19F NMR resonance shifts depending on the complementary nucleobase, providing more detailed information on Hg(II)-mediated base pairing.

Structural and Energetic Impact of Non-natural 7-Deaza-8-azaguanine, 7-Deaza-8-azaisoguanine, and Their 7-Substituted Derivatives on Hydrogen-Bond Pairing with Cytosine and Isocytosine.

The impact of 7-deaza-8-azaguanine (DAG) and 7-deaza-8-azaisoguanine (DAiG) modifications on the geometry and stability of the G:C Watson-Crick (cWW) base pair and the G:iC and iG:C reverse Watson-Crick (tWW) base pairs has been characterized theoretically. In addition, the effect on the same base pairs of seven C7-substituted DAG and DAiG derivatives, some of which have been previously experimentally characterized, has been investigated. Calculations indicate that all of these modifications have a negligible impact on the geometry of the above base pairs, and that modification of the heterocycle skeleton has a small impact on the base-pair interaction energies. Instead, base-pair interaction energies are dependent on the nature of the C7 substituent. For the 7-substituted DAG-C cWW systems, a linear correlation between the base-pair interaction energy and the Hammett constant of the 7-substituent is found, with higher interaction energies corresponding to more electron-withdrawing substituents. Therefore, the explored modifications are expected to be accommodated in both parallel and antiparallel nucleic acid duplexes without perturbing their geometry, while the strength of a base pair (and duplex) featuring a DAG modification can, in principle, be tuned by incorporating different substituents at the C7 position.

The influence of 5-fluorouracil anticancer drug on the DNA base pairs; a quantum chemical study.

In the present study, various hydrogen bonded complexes between five-fluorouracil (FU) with AT and GC base pairs were studied. First, to determine the affinity of different sites of the parent structures (FU, AT, and GC) for the hydrogen bond formation, their molecular electrostatic potentials are explored. The complexation energies and the strength of individual HBs of the plausible complexes were evaluated by energetic, geometric, spectroscopic, topologic, and molecular orbital descriptors. Our results reveal that, the FU-GC complexes (34.76-44.42 kcal mol-1) are more stable than the FU-AT ones (21.00-30.35 kcal mol-1). Among the complexes, the FU3-AT1 and FU3-GC3 are the most stable structures in the both series, which can be related to the sites with the highest affinity. Second, a detail analysis of the hydrogen bond descriptors were performed to elucidate the effect of FU on the strength of HB interactions within the base pairs. Interestingly, due to the formation of various interactions between the active sites of basic molecules, the strength of HB within the base pairs in the most cases increase about +2.75 and +.57 kcal mol-1 for the GC and AT nucleobases, respectively. Finally, several aromatic indices (HOMA, FLU, NICS (0) and NICS (1)) were applied to evaluate the significance of π-electron delocalization (π-ED) of 5/6 membered rings. These results clearly show that the π-ED of the benchmark systems increase with the formation or strengthening of the HB, which is in line with the resonance assisted hydrogen bond theory.

Genetically encoded RNA-based sensors for intracellular imaging of silver ions.

Silver has been widely used for disinfection. The cellular accumulation of silver ions (Ag+) is critical in these antibacterial effects. The direct cellular measurement of Ag+ is useful for the study of disinfection mechanisms. Herein, we reported a novel genetically encoded RNA-based sensor to image Ag+ in live bacterial cells. The sensor is designed by introducing a cytosine-Ag+-cytosine metallo base pair into a fluorogenic RNA aptamer, Broccoli. The binding of Ag+ induces the folding of Broccoli and activates a fluorescence signal. This sensor can be genetically encoded to measure the cellular flux and antibacterial effect of Ag+.

Structural alteration of low pH, low temperature induced protonated form of DNA to the canonical form by the benzophenanthridine alkaloid nitidine: Spectroscopic exploration.

Polymorphism of DNA plays a very important part of research relating to the drug-DNA interactions. Here main focus of our investigation is to monitor the interaction of the benzophenanthridine plant alkaloid, nitidine (NIT) with two different forms of DNA i.e. B-DNA and protonated form of DNA maintaining proper temperatures and buffer conditions. Binding interaction of NIT was ascertained from the UV-Visible spectroscopic and spectrofluorimetric titration experiments. Binding constants of the interactions of NIT with different polymorphic forms were calculated from UV-absorption study. The binding constants were 3.8 × 105 M-1 and 1.3 × 105 M-1 for B-DNA and protonated DNA respectively. Red shift in the absorption maxima of NIT on binding with DNA, comparatively greater relative quenching of fluorescence intensity of free NIT than bound NIT, perturbation in the CD spectrum of DNA in presence of NIT confirmed the mode of binding as intercalation. Moreover, spectropolarimetric experiment confirms that left handed protonated form of DNA gets partially converted to the canonical B form of DNA while binds with NIT. Besides the CD experiment, thermal melting experiment also showed that on binding with NIT stabilization of protonated DNA was increased to an appreciable extent.

Analysis of Interactions between Telomeric i-Motif DNA and a Cyclic Tetraoxazole Compound.

The interaction of a macrocyclic tetraoxazole compound, L2H2-4OTD (1), with two aminoalkyl side chains and telomeric i-motif, was investigated by means of electrophoretic mobility shift assay, circular dichroism spectroscopy, mass spectrometry and NMR spectroscopy analyses. The results indicate that 1 interacts with the i-motif structure at two preferred binding sites.

TAMRA-polypyrrole for A/T sequence visualization on DNA molecules.

Fluorophore-linked, sequence-specific DNA binding reagents can visualize sequence information on a large DNA molecule. In this paper, we synthesized newly designed TAMRA-linked polypyrrole to visualize adenine and thymine base pairs. A fluorescent image of the stained DNA molecule generates an intensity profile based on A/T frequency, revealing a characteristic sequence composition pattern. Computer-aided comparison of this intensity pattern with the genome sequence allowed us to determine the DNA sequence on a visualized DNA molecule from possible intensity profile pattern candidates for a given genome. Moreover, TAMRA-polypyrrole offers robust advantages for single DNA molecule detection: no fluorophore-mediated photocleavage and no structural deformation, since it exhibits a sequence-specific pattern alone without the use of intercalating dyes such as YOYO-1. Accordingly, we were able to identify genomic DNA fragments from Escherichia coli cells by aligning them to the genomic A/T frequency map based on TAMRA-polypyrrole-generated intensity profiles. Furthermore, we showed band and interband patterns of polytene chromosomal DNA stained with TAMRA-polypyrrole because it prefers to bind AT base pairs.

Novel pathway for mutagenic tautomerization of classical А∙Т DNA base pairs via sequential proton transfer through quasi-orthogonal transition states: A QM/QTAIM investigation.

In this paper we have theoretically predicted a novel pathway for the mutagenic tautomerization of the classical A∙T DNA base pairs in the free state, the Watson-Crick A·Ð¢(WC), reverse Watson-Crick A·Ð¢(rWC), Hoogsteen A·Ð¢(H) and reverse Hoogsteen A·Ð¢(rH) pairs, via sequential proton transfer accompanied by a significant change in the mutual orientation of the bases. Quantum-mechanical (QM) calculations were performed at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level in vacuum phase, along with Bader's quantum theory of Atoms in Molecules (QTAIM). These processes involve transition states (TSs) with quasi-orthogonal structures (symmetry C1), which are highly polar, tight ion pairs (A-, N6H2-deprotonated)∙(T+, O4/O2-protonated). Gibbs free energies of activation for the A∙T(WC) / A∙T(rWC) ↔ A*∙Т(rwWC) / A*∙Т(wWC) tautomeric transitions (~43.5 kcal∙mol-1) are lower than for the A∙T(H) / A∙T(rH) ↔ A*N7∙Т(rwH) / A*N7∙Т(wH) tautomerisations (~53.0 kcal∙mol-1) (rare tautomers are marked by an asterisk; w-wobble configured tautomerisation products). The (T)N3+H⋯N1-(A), (T)O4+H⋯N1-(A) / (T)N3+H⋯N1-(A) and (T)O2+H⋯N1-(A) H-bonds are found in the transition states TSA-·T+A·T(WC)↔A*·T(rwWC) / TSA-·T+A·T(rWC)↔A*·T(wWC). However, in the transition state TSA-·T+A·Ð¢(H)↔A*N7·T(rwH) / TSA-·T+A·Ð¢(rH)↔A*N7·T(wH), the (T)N3+H⋯N7-(A), (T)O4+H⋯N7-(A) / (T)N3+H⋯N7-(A) and (T)O2+H⋯N7-(A) H-bonds are supplemented by the attractive (T)O4+/O2+⋯N6-(A) van der Waals contacts. It was demonstrated that the products of the tautomerization of the classical A∙T DNA base pairs-A*∙Т(rwWC), A*N7∙Т(rwH) and A*N7∙Т(wH) (symmetry Cs)-further transform via double proton transfer into the energetically favorable wobble A∙T*(rwWC), A∙T*(rwH) and A∙T*O2(wH) base mispairs (symmetry Cs).

Role of 6-Mercaptopurine in the potential therapeutic targets DNA base pairs and G-quadruplex DNA: insights from quantum chemical and molecular dynamics simulations.

The theoretical studies on DNA with the anticancer drug 6-Mercaptopurine (6-MP) are investigated using theoretical methods to shed light on drug designing. Among the DNA base pairs considered, 6-MP is stacked with GC with the highest interaction energy of -46.19 kcal/mol. Structural parameters revealed that structure of the DNA base pairs is deviated from the planarity of the equilibrium position due to the formation of hydrogen bonds and stacking interactions with 6-MP. These deviations are verified through the systematic comparison between X-H bond contraction and elongation and the associated blue shift and red shift values by both NBO analysis and vibrational analysis. Bent's rule is verified for the C-H bond contraction in the 6-MP interacted base pairs. The AIM results disclose that the higher values of electron density (ρ) and Laplacian of electron density (∇2ρ) indicate the increased overlap between the orbitals that represent the strong interaction and positive values of the total electron density show the closed-shell interaction. The relative sensitivity of the chemical shift values for the DNA base pairs with 6-MP is investigated to confirm the hydrogen bond strength. Molecular dynamics simulation studies of G-quadruplex DNA d(TGGGGT)4 with 6-MP revealed that the incorporation of 6-MP appears to cause local distortions and destabilize the G-quadruplex DNA.

Effects of osmolytes on stable UUCG tetraloops and their preference for a CG closing base pair.

Osmolytes have the potential to affect the stability of secondary structure motifs and alter preferences for conserved nucleic acid sequences in the cell. To contribute to the understanding of the in vivo function of RNA we observed the effects of different classes of osmolytes on the UNCG tetraloop motif. UNCG tetraloops are the most common and stable of the RNA tetraloops and are nucleation sites for RNA folding. They also have a significant thermodynamic preference for a CG closing base pair. The thermal denaturation of model hairpins containing UUCG loops was monitored using UV-Vis spectroscopy in the presence of osmolytes with different chemical properties. Interestingly, all of the osmolytes tested destabilized the hairpins, but all had little effect on the thermodynamic preference for a CG base pair, except for polyethylene glycol (PEG) 200. PEG 200 destabilized the loop with the CG closing base pair relative to the loop with a GC closing base pair. The destabilization was linear with increasing concentrations of PEG 200, and the slope of this relationship was not perturbed by changes in the hairpin stem outside of the closing pair. This result suggests that in the presence of PEG 200, the UUCG loop with a GC closing base pair may retain some preferential interactions with the cosolute that are lost in the presence of the CG closing base pair. These results reveal that relatively small structural changes may influence how osmolytes tune the stability, and thus the function of a secondary structure motif in vivo.

Robust IR-based detection of stable and fractionally populated G-C+ and A-T Hoogsteen base pairs in duplex DNA.

Noncanonical G-C+ and A-T Hoogsteen base pairs can form in duplex DNA and play roles in recognition, damage repair, and replication. Identifying Hoogsteen base pairs in DNA duplexes remains challenging due to difficulties in resolving syn versus antipurine bases with X-ray crystallography; and size limitations and line broadening can make them difficult to characterize by NMR spectroscopy. Here, we show how infrared (IR) spectroscopy can identify G-C+ and A-T Hoogsteen base pairs in duplex DNA across a range of different structural contexts. The utility of IR-based detection of Hoogsteen base pairs is demonstrated by characterizing the first example of adjacent A-T and G-C+ Hoogsteen base pairs in a DNA duplex where severe broadening complicates detection with NMR.

Assessment of new triplet forming artificial nucleobases as RNA ligands directed towards HCV IRES IIId loop.

We report the synthesis of two new artificial nucleobase scaffolds, 1 and 2, featuring adequate hydrogen bonding donors and acceptors for the molecular recognition of U:A and C:G base pairs, respectively. The tethering of these structures to various amino acids and the assessment of these artificial nucleobase-amino acid conjugates as RNA ligands against a model of HCV IRES IIId domain are also reported. Compound 1e displayed the highest affinity (Kd twice lower than neomycin - control). Moreover, it appears that this interaction is enthalpically and entropically favored.

Mutagen Synergy: Hypermutability Generated by Specific Pairs of Base Analogs.

UNLABELLED: We tested pairwise combinations of classical base analog mutagens in Escherichia coli to study possible mutagen synergies. We examined the cytidine analogs zebularine (ZEB) and 5-azacytidine (5AZ), the adenine analog 2-aminopurine (2AP), and the uridine/thymidine analog 5-bromodeoxyuridine (5BrdU). We detected a striking synergy with the 2AP plus ZEB combination, resulting in hypermutability, a 35-fold increase in mutation frequency (to 53,000 × 10(-8)) in the rpoB gene over that with either mutagen alone. A weak synergy was also detected with 2AP plus 5AZ and with 5BrdU plus ZEB. The pairing of 2AP and 5BrdU resulted in suppression, lowering the mutation frequency of 5BrdU alone by 6.5-fold. Sequencing the mutations from the 2AP plus ZEB combination showed the predominance of two new hot spots for A·T→G·C transitions that are not well represented in either single mutagen spectrum, and one of which is not found even in the spectrum of a mismatch repair-deficient strain. The strong synergy between 2AP and ZEB could be explained by changes in the dinucleoside triphosphate (dNTP) pools. IMPORTANCE: Although mutagens have been widely studied, the mutagenic effects of combinations of mutagens have not been fully researched. Here, we show that certain pairwise combinations of base analog mutagens display synergy or suppression. In particular, the combination of 2-aminopurine and zebularine, analogs of adenine and cytidine, respectively, shows a 35-fold increased mutation frequency compared with that of either mutagen alone. Understanding the mechanism of synergy can lead to increased understanding of mutagenic processes. As combinations of base analogs are used in certain chemotherapy regimens, including those involving ZEB and 5AZ, these results indicate that testing the mutagenicity of all drug combinations is prudent.

How the intercalation of phenanthroline affects the structure, energetics, and bond properties of DNA base pairs: theoretical study applied to adenine-thymine and guanine-cytosine tetramers.

The effects of phenanthroline (phen) intercalation on the structure, energetics, and bonding of adenine-thymine and guanine-cytosine tetramers (A-T/T-A and G-C/C-G) were studied through density functional theory (DFT) using functionals that were recently improved to consider the effect of dispersion forces. Our results given by energy decomposition analysis show that the dispersion contribution, ΔEdisp, is the most important contribution to the interaction energy, ΔEint. However, it is not enough to compensate the Pauli repulsion term, ΔEPauli, and the roles of the orbital contribution, ΔEorb, and, in particular, the electrostatic contribution, ΔEelstat, become crucial for the stabilization of the structures in the intercalation process. On the other hand, for G-C/C-G systems, hydrogen-bonding (HB) interactions are more important than stacking (S) interactions, whereas for A-T/T-A systems, HB and S become competitive. Moreover, intercalation produces important changes not only in the hydrogen bonds of base pairs, because S and HB are deeply connected, but also in other characteristic geometric parameters of the base pairs.