Complex Biophysical and Computational Analyses of G-Quadruplex Ligands: The Porphyrin Stacks Back.

G-quadruplexes (G4 s), as non-canonical DNA structures, attract a great deal of research interest in the molecular biology as well as in the material science fields. The use of small molecules as ligands for G-quadruplexes has emerged as a tool to regulate gene expression and telomeres maintenance. Meso-tetrakis-(N-methyl-4-pyridyl) porphyrin (TMPyP4) was shown as one of the first ligands for G-quadruplexes and it is still widely used.

Dimeric structures of DNA ATTTC repeats promoted by divalent cations.

Structural studies of repetitive DNA sequences may provide insights why and how certain repeat instabilities in their number and nucleotide sequence are managed or even required for normal cell physiology, while genomic variability associated with repeat expansions may also be disease-causing. The pentanucleotide ATTTC repeats occur in hundreds of genes important for various cellular processes, while their insertion and expansion in noncoding regions are associated with neurodegeneration, particularly with subtypes of spinocerebellar ataxia and familial adult myoclonic epilepsy. We describe a new striking domain-swapped DNA-DNA interaction triggered by the addition of divalent cations, including Mg2+ and Ca2+.

In-Cell Stability Prediction of RNA/DNA Hybrid Duplexes for Designing Oligonucleotides Aimed at Therapeutics.

In cells, the formation of RNA/DNA hybrid duplexes regulates gene expression and modification. The environment inside cellular organelles is heterogeneously crowded with high concentrations of biomolecules that affect the structure and stability of RNA/DNA hybrid duplexes. However, the detailed environmental effects remain unclear.

A Screening Protocol for Exploring Loop Length Requirements for the Formation of a Three Cytosine-Cytosine Base-Paired i-Motif.

DNA sequences containing at least four runs of repetitive cytosines can fold into tetra-helical structures called i-Motifs (iMs). The interest in these DNA secondary structures is increasing due to their therapeutical and technological applications. Still, limited knowledge of their folding requirements is currently available.

4'-SCF -Labeling Constitutes a Sensitive F NMR Probe for Characterization of Interactions in the Minor Groove of DNA.

Fluorinated nucleotides are invaluable for F NMR studies of nucleic acid structure and function. Here, we synthesized 4'-SCF -thymidine (T ) and incorporated it into DNA by means of solid-phase DNA synthesis. NMR studies showed that the 4'-SCF group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures.

Phen-DC Induces Refolding of Human Telomeric DNA into a Chair-Type Antiparallel G-Quadruplex through Ligand Intercalation.

Human telomeric G-quadruplex DNA structures are attractive anticancer drug targets, but the target's polymorphism complicates the drug design: different ligands prefer different folds, and very few complexes have been solved at high resolution. Here we report that Phen-DC , one of the most prominent G-quadruplex ligands in terms of high binding affinity and selectivity, causes dTAGGG(TTAGGG) to completely change its fold in KCl solution from a hybrid-1 to an antiparallel chair-type structure, wherein the ligand intercalates between a two-quartet unit and a pseudo-quartet, thereby ejecting one potassium ion. This unprecedented high-resolution NMR structure shows for the first time a true ligand intercalation into an intramolecular G-quadruplex.

On the interaction of an anticancer trisubstituted naphthalene diimide with G-quadruplexes of different topologies: a structural insight.

Naphthalene diimides showed significant anticancer activity in animal models, with therapeutic potential related to their ability to strongly interact with G-quadruplexes. Recently, a trifunctionalized naphthalene diimide, named NDI-5, was identified as the best analogue of a mini-library of novel naphthalene diimides for its high G-quadruplex binding affinity along with marked, selective anticancer activity, emerging as promising candidate drug for in vivo studies. Here we used NMR, dynamic light scattering, circular dichroism and fluorescence analyses to investigate the interactions of NDI-5 with G-quadruplexes featuring either parallel or hybrid topology.

4'-Fluorinated RNA: Synthesis, Structure, and Applications as a Sensitive F NMR Probe of RNA Structure and Function.

Fluorinated RNA molecules, particularly 2'-F RNA, have found a wide range of applications in RNA therapeutics, RNA aptamers, and ribozymes and as F NMR probes for elucidating RNA structure. Owing to the instability of 4'-F ribonucleosides, synthesis of 4'-F-modified RNA has long been a challenge. In this study, we developed a strategy for synthesizing a 4'-F-uridine (U) phosphoramidite, and we used it to prepare 4'-F RNA successfully.

Chasing Particularities of Guanine- and Cytosine-Rich DNA Strands.

By substitution of natural nucleotides by their abasic analogs (i.e., 1',2'-dideoxyribose phosphate residue) at critically chosen positions within 27-bp DNA constructs originating from the first intron of gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures.

G-quadruplex-mediated reduction of a pathogenic mitochondrial heteroplasmy.

Disease-associated variants in mitochondrial DNA (mtDNA) are frequently heteroplasmic, a state of co-existence with the wild-type genome. Because heteroplasmy correlates with the severity and penetrance of disease, improvement in the ratio between these genomes in favor of the wild-type, known as heteroplasmy shifting, is potentially therapeutic. We evaluated known pathogenic mtDNA variants and identified those with the potential for allele-specific differences in the formation of non-Watson-Crick G-quadruplex (GQ) structures.

Effect of Ribose Conformation on RNA Cleavage via Internal Transesterification.

RNA cleavage via internal transesterification is a fundamental reaction involved in RNA processing and metabolism, and the regulation thereof. Herein, the influence of ribose conformation on this reaction was investigated with conformationally constrained ribonucleotides. RNA cleavage rates were found to decrease in the order South-constrained ribonucleotide > native ribonucleotide ≫ North-constrained counterpart, indicating that the ribose conformation plays an important role in modulating RNA cleavage via internal transesterification.

Pursuing origins of (poly)ethylene glycol-induced G-quadruplex structural modulations.

Molecular crowding conditions provided by high concentration of cosolutes are utilized for characterization of biomolecules in cell-mimicking environment and development of drug-delivery systems. In this context, (poly)ethylene glycols are often used for studying non-canonical DNA structures termed G-quadruplexes, which came into focus by emerging structural biology findings and new therapeutic drug design approaches. Recently, several reports were made arguing against using (poly)ethylene glycols in role of molecular crowding agents due to their direct impact on DNA G-quadruplex stability and topology.

Interactions of Pt-ttpy with G-Quadruplexes Originating from Promoter Region of the c-myc Gene Deciphered by NMR and Gel Electrophoresis Analysis.

This study provides insights into the interactions of Pt-ttpy, that is, a metallo-organic heterocycle-comprising platinum(II) complex of terpyridine, and G-quadruplexes adopted by G-rich DNA from the transcriptional regulatory element of the c-myc gene, a well-known attractive target for artificial modulation of oncogene expression. A previously noted drug-like potential of Pt-ttpy relies on its antiproliferative activity on cancer cells and its increased selectivity for G-quadruplex binding attributed to the combination of distinct interacting modes. The predominant interaction between the herein used models of a parallel G-quadruplex exhibiting short propeller-type loops and Pt-ttpy occurs through stacking to the outer G-quartets.

Unique structural features of interconverting monomeric and dimeric G-quadruplexes adopted by a sequence from the intron of the N-myc gene.

A multidimensional heteronuclear NMR study has demonstrated that a guanine-rich DNA oligonucleotide originating from the N-myc gene folds into G-quadruplex structures in the presence of K(+), NH(4)(+), and Na(+) ions. A monomeric G-quadruplex formed in K(+) ion containing solution exhibits three G-quartets and flexible propeller-type loops. The 3D structure with three single nucleotide loops represents a missing element in structures of parallel G-quadruplexes.

Cation localization and movement within DNA thrombin binding aptamer in solution.

The thrombin binding aptamer, 15-mer oligonucleotide d[G(2)T(2)G(2)TGTG(2)T(2)G(2)], was folded into the well known antiparallel unimolecular G-quadruplex in the presence of (15)NH(4)(+) ions. Although the formed G-quadruplex is thermodynamically less stable than in the presence of K(+) ions, the loop conformations and folding topology are the same. On the other hand, titration of Na(+) ions into an aqueous solution of TBA resulted in the formation of one major and several minor species of G-quadruplexes.