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+. The results of NMR characterization of d(ATTTC)3 in solution show that the oligonucleotide folds into a novel 3D architecture with two central C:C+ base pairs sandwiched between a couple of T:T base pairs. This structural element, referred to here as the TCCTzip, is characterized by intercalative hydrogen-bonding, while the nucleobase moieties are poorly stacked. The 5'- and 3'-ends of TCCTzip motif are connected by stem-loop segments characterized by A:T base pairs and stacking interactions. Insights embodied in the non-canonical DNA structure are expected to advance our understanding of why only certain pyrimidine-rich DNA repeats appear to be pathogenic, while others can occur in the human genome without any harmful consequences.

Structure of native four-repeat satellite III sequence with non-canonical base interactions.

Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5'-ATGGA ATGGA ATGGA ATGGA-3' using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05-0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G-G intercalation sandwiched by sheared A-G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A-T/T-A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

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. Therefore, the mechanistic details of the effect of such molecular crowding were investigated at the molecular level by using thermodynamic and nuclear magnetic resonance analyses, revealing structure-dependent destabilization of the duplexes under crowded conditions. The transition from B- to A-like hybrid duplexes due to a change in conformation of the DNA strand guided by purine-pyrimidine asymmetry significantly increased the hydration number, which resulted in greater destabilization by the addition of cosolutes. By quantifying the individual contributions of environmental factors and the bulk structure of the duplex, we developed a set of parameters that predict the stability of hybrid duplexes with conformational dissimilarities under diverse crowding conditions. A comparison of the effects of environmental conditions in living cells and in vitro crowded solutions on hybrid duplex formation using the Förster resonance energy transfer technique established the applicability of our parameters to living cells. Moreover, our derived parameters can be used to estimate the efficiency of transcriptional inhibition, genome editing, and silencing techniques in cells. This supports the usefulness of our parameters for the visualization of cellular mechanisms of gene expression and the development of nucleic acid-based therapeutics targeting different cells.

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. We developed a novel step-by-step pipeline for the systematic screening of putative iM-forming model sequences. Focusing on structures comprising only three cytosine-cytosine+ base pairs, we investigated what the minimal lengths of the loops required for formation of an intra-molecular iM are. Our data indicate that two and three nucleotides are required to connect the strands through the minor and majorgrooves of the iM, respectively. Additionally, they highlight an asymmetric behavior according to the distribution of the cytosines. Specifically, no sequence containing a single cytosine in the first and third run was able to fold into intra-molecular iMs with the same stability of those formed when the first and the third run comprise two cytosines. This knowledge represents a step forward toward the development of prediction tools for the proper identification of biologically functional iMs, as well as for the rational design of these secondary structures as technological devices.

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. Interplay of different binding modes of NDI-5 to G-quadruplexes was observed for both parallel and hybrid topologies, with end-stacking always operative as the predominant binding event. While NDI-5 primarily targets the 5'-end quartet of the hybrid G-quadruplex model (m-tel24), the binding to a parallel G-quadruplex model (M2) occurs seemingly simultaneously at the 5'- and 3'-end quartets. With parallel G-quadruplex M2, NDI-5 formed stable complexes with 1:3 DNA:ligand binding stoichiometry. Conversely, when interacting with hybrid G-quadruplex m-tel24, NDI-5 showed multiple binding poses on a single G-quadruplex unit and/or formed different complexes comprising two or more G-quadruplex units. NDI-5 produced stabilizing effects on both G-quadruplexes, forming complexes with dissociation constants in the nM range.

Phen-DC3 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-DC3 , one of the most prominent G-quadruplex ligands in terms of high binding affinity and selectivity, causes dTAGGG(TTAGGG)3 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.

4'-SCF3 -Labeling Constitutes a Sensitive 19 F NMR Probe for Characterization of Interactions in the Minor Groove of DNA.

Fluorinated nucleotides are invaluable for 19 F NMR studies of nucleic acid structure and function. Here, we synthesized 4'-SCF3 -thymidine (T 4 ' - SCF 3 ${{^{4{^\prime}\hbox{-}{\rm SCF}{_{3}}}}}$ ) and incorporated it into DNA by means of solid-phase DNA synthesis. NMR studies showed that the 4'-SCF3 group exhibited a flexible orientation in the minor groove of DNA duplexes and was well accommodated by various higher order DNA structures. The three magnetically equivalent fluorine atoms in 4'-SCF3 -DNA constitute an isolated spin system, offering high 19 F NMR sensitivity and excellent resolution of the positioning of T 4 ' - SCF 3 ${{^{4{^\prime}\hbox{-}{\rm SCF}{_{3}}}}}$ within various secondary and tertiary DNA structures. The high structural adaptability and high sensitivity of T 4 ' - SCF 3 ${{^{4{^\prime}\hbox{-}{\rm SCF}{_{3}}}}}$ make it a valuable 19 F NMR probe for quantitatively distinguishing diverse DNA structures with single-nucleotide resolution and for monitoring the dynamics of interactions in the minor groove of double-stranded DNA.

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. We found that the Leigh syndrome (LS)-associated m.10191C variant promotes GQ formation within local sequence in vitro. Interaction of this sequence with a small molecule GQ-binding agent, berberine hydrochloride, further increased GQ stability. The GQ formed at m.10191C differentially impeded the processivity of the mitochondrial DNA polymerase gamma (Pol γ) in vitro, providing a potential means to favor replication of the wild-type allele. We tested the potential for shifting heteroplasmy through the cyclical application of two different mitochondria-targeted GQ binding compounds in primary fibroblasts from patients with m.10191T>C heteroplasmy. Treatment induced alternating mtDNA depletion and repopulation and was effective in shifting heteroplasmy towards the non-pathogenic allele. Similar treatment of pathogenic heteroplasmies that do not affect GQ formation did not induce heteroplasmy shift. Following treatment, heteroplasmic m.10191T>C cells had persistent improvements and heteroplasmy and a corresponding increase in maximal mitochondrial oxygen consumption. This study demonstrates the potential for using small-molecule GQ-binding agents to induce genetic and functional improvements in m.10191T>C heteroplasmy.

4'-Fluorinated RNA: Synthesis, Structure, and Applications as a Sensitive 19F 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 19F 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 (4'FU) phosphoramidite, and we used it to prepare 4'-F RNA successfully. In the context of an RNA strand, 4'FU, which existed in a North conformation, was reasonably stable and resembled unmodified uridine well. The 19F NMR signal of 4'FU was sensitive to RNA secondary structure, with a chemical shift dispersion as large as 4 ppm (compared with <1 ppm for 2'FU), which makes it a valuable probe for discriminating single-stranded RNA and A-type, B-type, and mismatched duplexes. In addition, we demonstrated that because RNA-processing enzymes treated 4'FU the same as unmodified uridine, 4'FU could be used to monitor RNA structural dynamics and enzyme-mediated RNA processing. Taken together, our results indicate that 4'-F RNA represents a probe with wide utility for elucidation of RNA structure and function by means of 19F NMR spectroscopy.

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. However, the available data on structural details underlying DNA interaction is very scarce and thus limits in-depth comprehension. Herein, structural and thermodynamic analyses were strategically combined to assess G-quadruplex-cosolute interactions and address previously reported variances regarding the driving forces of G-rich DNA structural transformations under molecular crowding conditions. With the use of complementary (CD, NMR and UV) spectroscopic methods and model approach we characterized DNA G-quadruplex in the presence of the smallest and one of the largest typically used (poly)ethylene glycols. Dehydration effect is the key contributor to ethylene-glycol-induced increased stability of the G-quadruplex, which is in the case of the large cosolute mainly guided by the subtle direct interactions between PEG 8000 and the outer G-quartet regions.

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 N-myc gene, we hindered hybridization within the guanine- and cytosine-rich central region and followed formation of non-canonical structures. The impeded hybridization between the complementary strands leads to time-dependent structural transformations of guanine-rich strand that are herein characterized with the use of solution-state NMR, CD spectroscopy, and native polyacrylamide gel electrophoresis. Moreover, the DNA structural changes involve transformation of intra- into inter-molecular G-quadruplex structures that are thermodynamically favored. Intriguingly, the transition occurs in the presence of complementary cytosine-rich strands highlighting the inability of Watson-Crick base-pairing to preclude the transformation between G-quadruplex structures that occurs via intertwining mechanism and corroborates a role of G-quadruplex structures in DNA recombination processes.

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.

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. The presence of adenine versus thymine residue at the 5'-end overhanging region allows the coordinative binding of Pt-ttpy to the G-quadruplex structure. Interestingly, Pt-ttpy triggers the formation of the G-quadruplex even in the absence of cations. Furthermore, NMR-based characterisation revealed common structural features of Pt-ttpy-G-quadruplex complexes in the presence and absence of cations, which indicate that cations may be expelled from the cores of the corresponding structures.

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. The structural features together with the high temperature stability are suggestive of the specific biological role of G-quadruplex formation within the intron of the N-myc gene. An increase in K(+) ion and oligonucleotide concentrations resulted in transformation of the monomeric G-quadruplex into a dimeric form. The dimeric G-quadruplex exhibits six stacked G-quartets, parallel strand orientations, and propeller-type loops. A link between the third and the fourth G-quartets consists of two adenine residues that are flipped out to facilitate consecutive stacking of six G-quartets.

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. Solution-state NMR was used to localize (15)NH(4)(+) ions between the two G-quartets within the core of the structure, and to determine the equilibrium binding constant, which equals 190 M(-1). No other potential cation binding sites were resolved on the time-scale of NMR spectrometer. Exchange of (15)NH(4)(+) ions between the inner binding site and bulk solution is characterized by the exchange rate constant of 1.0 s(-1) at 15 degrees C. T4 and T13 form a noncanonical base pair, which greatly affects access of bulk ions into the cation binding site in the G-quadruplex core. G2 and G11 exhibit out of plane bending towards the two TT loops away from the bound (15)NH(4)(+) ions, which in turn exposes them to more efficient chemical exchange processes with bulk ions and water.