Quadruplexes Are Everywhere…On the Other Strand Too: The i-Motif

International audiencei-Motif (the name stems from “intercalated”), also known as i-DNA, is a pH-dependent four-stranded nucleic acid structure formed by cytosine-rich sequences via hemi-protonated and intercalated CC+ base pairs. Although this structure is favored at acidic pH, recent evidence has demonstrated its existence in vivo, stimulating the exploration of its biological roles. Before that, it was mostly regarded as a mere structural oddity, or a tool for bio- and nanotechnol- ogies: its unique pH-sensitive nature makes it a remarkable candidate as a nanodevice and pH sensor. In this chapter, we provide a general panorama of this structure. The history and basic knowledge of i-motif are provided first. Then, we present the main characterization methods of i-motif and factors affecting i-motif stability. Following that, we focus on the applications of i-motif in nanotechnology and analytical chemistry. Last, the interaction between i-motif and ligands and the physiological roles of i-motif are briefly introduced. We argue that the i-motif, similar to its complementary G-quadruplex, is an attractive structure for multidisciplinary approaches. It serves as a basic component for various applications and has been proposed to play biological roles in vivo

Quadruplexes Are Everywhere…On the Other Strand Too: The i-Motif

International audiencei-Motif (the name stems from “intercalated”), also known as i-DNA, is a pH-dependent four-stranded nucleic acid structure formed by cytosine-rich sequences via hemi-protonated and intercalated CC+ base pairs. Although this structure is favored at acidic pH, recent evidence has demonstrated its existence in vivo, stimulating the exploration of its biological roles. Before that, it was mostly regarded as a mere structural oddity, or a tool for bio- and nanotechnol- ogies: its unique pH-sensitive nature makes it a remarkable candidate as a nanodevice and pH sensor. In this chapter, we provide a general panorama of this structure. The history and basic knowledge of i-motif are provided first. Then, we present the main characterization methods of i-motif and factors affecting i-motif stability. Following that, we focus on the applications of i-motif in nanotechnology and analytical chemistry. Last, the interaction between i-motif and ligands and the physiological roles of i-motif are briefly introduced. We argue that the i-motif, similar to its complementary G-quadruplex, is an attractive structure for multidisciplinary approaches. It serves as a basic component for various applications and has been proposed to play biological roles in vivo

The noncovalent dimerization of a G-quadruplex/hemin DNAzyme improves its biocatalytic properties

International audienceWhile many protein enzymes exert their functions through multimerization, which improves both selectivity and activity, this has not yet been demonstrated for other naturally occurring catalysts. Here, we report on a multimerization effect applied to catalytic DNAs (or DNAzymes) and demonstrate that the enzymatic proficiency of G-quadruplexes (GQs) in interaction with hemin cofactor is remarkably enhanced by homodimerization. The resulting non-covalent dimeric GQ-DNAzyme system provides hemin with a structurally defined active site in which both the cofactor (hemin) and the oxidant (H2O2) are activated. This new biocatalytic system efficiently performs peroxidase-and peroxygenase-type biotransformations of a broad range of substrates, thus opening new perspectives for the biotechnological applications of GQs

Fluorescence Spectroscopic Insight into the Supramolecular Interactions in DNA-Based Enantioselective Sulfoxidation

Interactions of copper(II)-bipyridine cofactors and thioanisole substrate with human telomeric G-quadruplex DNA were studied by UV/Vis absorption, circular dichroism, and fluorescence quenching titration. Three copper(II)-bipyridine complexes are equivalently anchored to the G-quadruplex scaffold at all five fluorescently labeled sites. Thioanisole interacts with the DNA architecture at both the second loop and 3 terminus in the absence or presence of copper(II)-bipyridine complexes. These nonspecificities in the weak interactions of Cu-II complexes and thioanisole with G-quadruplex might explain why DNA only affords a modest enantioselectivity in the oxidation of thioanisole. These findings provide insights toward the construction of highly enantioselective DNA-based catalysts

Probing the interaction of copper cofactor and azachalcone substrate with G-quadruplex of DNA based Diels-Alderase by site-specific fluorescence quenching titration

DNAzymes have been widely used in biosensors, asymmetric synthesis and pharmaceuticals. Typically, metal cofactor and substrate interact with DNA by supramolecular interactions in DNAzyme based asymmetric catalysis. However, binding positions of cofactor and substrate with DNA scaffold are not well understood, which is an obstacle to reveal the assembly and catalysis mechanisms of DNAzyme. Herein, we report a method of site-specific fluorescence quenching titration to elucidate the assembly and catalysis processes of a G-quadruplex based Diels-Alderase DNAzyme. Titration data indicate that cofactor Cu(II)-terpyridine stacked atop 5' and 3' external G-quartets with high and low binding affinities respectively, and induced the G-quadruplex to form a hybrid-1 topology. Substrate azachalcone interacted with 3' quartet exclusively, implicating that asymmetric Diels-Alder cycloaddition may occur at 3' G-quartet. In addition, enzyme kinetic analyses show that activity and enantioselectivity of the DNAzyme were substantially preserved after attaching the fluorophores. Overall, site-specific fluorescence quenching is a concise and efficient approach to probe the assembly processes of DNAzyme. (c) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved

Drivers of i-DNA Formation in a Variety of Environments Revealed by Four-Dimensional UV Melting and Annealing

International audiencei-DNA is a four-stranded, pH-sensitive structure formed by cytosine-rich DNA sequences. Previous reports have addressed the conditions for formation of this motif in DNA in vitro and validated its existence in human cells. Unfortunately, these in vitro studies have often been performed under different experimental conditions, making comparisons difficult. To overcome this, we developed a four-dimensional UV melting and annealing (4DUVMA) approach to analyze i-DNA formation under a variety of conditions (e.g., pH, temperature, salt, crowding). Analysis of 25 sequences provided a global understanding of i-DNA formation under disparate conditions, which should ultimately allow the design of accurate prediction tools. For example, we found reliable linear correlations between the mid-point of pH transition and temperature (-0.04 ± 0.003 pH unit per 1.0 °C temperature increment) and between the melting temperature and pH (-23.8 ± 1.1 °C per pH unit increment). In addition, by analyzing the hysteresis between denaturing and renaturing profiles in both pH and thermal transitions, we found that loop length, nature of the C-tracts, pH, temperature, and crowding agents all play roles in i-DNA folding kinetics. Interestingly, our data indicate which conformer is more favorable for the sequences with an odd number of cytosine base pairs. Then the h m l pH l f " "-DNAs from human promoter genes were measured under near physiological conditions (pH 7.0, 37 °C). The 4DUVMA method can become a universal resource to analysis the properties of any i-DNA-prone sequence

Higher-Order Human Telomeric G-Quadruplex DNA Metalloenzymes Enhance Enantioselectivity in the Diels-Alder Reaction

Short human telomeric (HT) DNA sequences form single G-quadruplex (G(4)) units and exhibit structure-based stereocontrol for a series of reactions. However, for more biologically relevant higher-order HT G(4)-DNAs (beyond a single G(4) unit), the catalytic performances are unknown. Here, we found that higher-order HT G(4)-DNA copper metalloenzymes (two or three G(4) units) afford remarkably higher enantioselectivity (>90% ee) and a five- to sixfold rate increase, compared to a single G(4) unit, for the Diels-Alder reaction. Electron paramagnetic resonance (EPR) and enzymatic kinetic studies revealed that the distinct catalytic function between single and higher-order G(4)-DNA copper metalloenzymes can be attributed to different Cu-II coordination environments and substrate specificity. Our finding suggests that, like protein enzymes and ribozymes, higher-order structural organization is crucial for G(4)-DNA-based catalysis

Loop permutation affects the topology and stability of G-quadruplexes

G-quadruplexes are unusual DNA and RNA secondary structures ubiquitous in a variety of organisms including vertebrates, plants, viruses and bacteria. The folding topology and stability of intramolecular G-quadruplexes are determined to a large extent by their loops. Loop permutation is defined as swapping two or three of these regions so that intramolecular G-quadruplexes only differ in the sequential order of their loops. Over the past two decades, both length and base composition of loops have been studied extensively, but a systematic study on the effect of loop permutation has been missing. In the present work, 99 sequences from 21 groups with different loop permutations were tested. To our surprise, both conformation and thermal stability are greatly dependent on loop permutation. Loop permutation actually matters as much as loop length and base composition on G-quadruplex folding, with effects on T-m as high as 17 degrees C. Sequences containing a longer central loop have a high propensity to adopt a stable non-parallel topology. Conversely, sequences containing a short central loop tend to form a parallel topology of lower stability. In addition, over half of interrogated sequences were found in the genomes of diverse organisms, implicating their potential regulatory roles in the genome or as therapeutic targets. This study illustrates the structural roles of loops in G-quadruplex folding and should help to establish rules to predict the folding pattern and stability of G-quadruplexes

How Proximal Nucleobases Regulate the Catalytic Activity of G-Quadruplex/Hemin DNAzymes

International audienceG-quadruplexes (G4s) are versatile catalytic DNAs when combined with hemin. Despite the repertoire of catalytically competent G4/hemin complexes studied so far, little is known about the detailed catalytic mechanism of these biocatalysts. Herein, we have carried out an in-depth analysis of the hemin binding site within the G4/hemin catalysts, providing the porphyrinic cofactor with a controlled nucleotidic environment. We intensively assessed the position-dependent catalytic enhancement in model reactions and found that proximal nucleobases enhance the catalytic ability of the G4/hemin complexes. Our results allow for revisiting the mechanism of the G4/hemin-based catalysis, especially gaining insights into the rate-limiting step, demonstrating how both the G4 core and the proximal nucleotides dA and/or dC concomitantly activate the Compound 0 → 0* prototropic cleavage of H 2 O 2 to foster Compound 1 formation. These results provide mechanistic clues as to how the properties of G4-based catalysts can be improved to ultimately make them competitive with proteinaceous enzymes

Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme

The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence, of 100 mM K+, loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. D-1 NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme