Chimeric Biocatalyst Combining Peptidic and Nucleic Acid Components Overcomes the Performance and Limitations of the Native Horseradish Peroxidase.

Chimeric peptide-DNAzyme (CPDzyme) is a novel artificial peroxidase that relies on the covalent assembly of DNA, peptides, and an enzyme cofactor in a single scaffold. An accurate control of the assembly of these different partners allows for the design of the CPDzyme prototype G4-Hemin-KHRRH, found to be >2000-fold more active (in terms of conversion number kcat) than the corresponding but non-covalent G4/Hemin complex and, more importantly, >1.5-fold more active than the corresponding native peroxidase (horseradish peroxidase) when considering a single catalytic center. This unique performance originates in a series of gradual improvements, thanks to an accurate selection and arrangement of the different components of the CPDzyme, in order to benefit from synergistic interactions between them. The optimized prototype G4-Hemin-KHRRH is efficient and robust as it can be used under a wide range of non-physiologically relevant conditions [organic solvents, high temperature (95 °C), and in a wide range of pH (from 2 to 10)], thus compensating for the shortcomings of the natural enzymes. Our approach thus opens broad prospects for the design of ever more efficient artificial enzymes.

DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence.

BACKGROUND: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. RESULTS: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. CONCLUSIONS: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies.

The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology.

Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the 'flanking effect' in this work. This 'flanking effect' was more pronounced when nucleotides were added at the 5'-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5'-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context.

Genome wide distribution of G-quadruplexes and their impact on gene expression in malaria parasites.

Mechanisms of transcriptional control in malaria parasites are still not fully understood. The positioning patterns of G-quadruplex (G4) DNA motifs in the parasite's AT-rich genome, especially within the var gene family which encodes virulence factors, and in the vicinity of recombination hotspots, points towards a possible regulatory role of G4 in gene expression and genome stability. Here, we carried out the most comprehensive genome-wide survey, to date, of G4s in the Plasmodium falciparum genome using G4Hunter, which identifies G4 forming sequences (G4FS) considering their G-richness and G-skewness. We show an enrichment of G4FS in nucleosome-depleted regions and in the first exon of var genes, a pattern that is conserved within the closely related Laverania Plasmodium parasites. Under G4-stabilizing conditions, i.e., following treatment with pyridostatin (a high affinity G4 ligand), we show that a bona fide G4 found in the non-coding strand of var promoters modulates reporter gene expression. Furthermore, transcriptional profiling of pyridostatin-treated parasites, shows large scale perturbations, with deregulation affecting for instance the ApiAP2 family of transcription factors and genes involved in ribosome biogenesis. Overall, our study highlights G4s as important DNA secondary structures with a role in Plasmodium gene expression regulation, sub-telomeric recombination and var gene biology.

Highly Sensitive Biosensing Applications of a Magnetically Immobilizable Covalent G-Quadruplex-Hemin DNAzyme Catalytic System.

G-quadruplex/hemin (G4/hemin) DNAzymes are biosensing systems, but their application remains limited by an overall low activity and a rather high level of unwarranted background reactions. Here, these issues were addressed through the rational design of F3T-azaC-hemin, a G4-based construct in which the hemin is covalently linked to the G4 core and its binding site flanked with a nucleotide activator, here d(T-azaC). This design led to a G4-DNAzyme whose performances have been ca. 150-fold increased compared to the parent G4-based system. The utility of F3T-azaC-hemin was demonstrated here through the ultrasensitive chemiluminescent detection of miRNA-221. The limit of detection (LOD) has been decreased to the femtomolar range, making it a new and highly efficient molecular tool in the biosensing technology field.

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

i-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 midpoint 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 thermal and pH stabilities of "native" i-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 analyze the properties of any i-DNA-prone sequence.

Highly Selective Detection of K+ Based on a Dimerized G-Quadruplex DNAzyme.

Potassium ion (K+) plays a crucial role in biological systems, such as maintaining cellular processes and causing diseases. However, specifically, the detection of K+ is extremely challenging because of the coexistence of the chemically similar ion of Na+ under physiological conditions. In this work, a K+ specific biosensor is constructed on the basis of a dimerized G-quadruplex (GQ) DNA, which is promoted by K+, and the enzymatic activity of the resulting DNAzyme depends on the concentration of the K+. The K+ in a 1-200 mM concentration range can be selectively detected by visual color, UV-Vis absorbance or fluorescence even if the concentration of the accompanying Na+ is up to 140 mM at an ambient condition up to 45 °C. In addition, this system can also be used to selectively detect NH4+ in a 5-200 mM concentration range. This dimerized DNAzyme offers a new type of biosensor with a potential application in the biological system.

Effects of Sequence and Base Composition on the CD and TDS Profiles of i-DNA.

The i-motif DNA, also known as i-DNA, is a non-canonical DNA secondary structure formed by cytosine-rich sequences, consisting of two intercalated parallel-stranded duplexes held together by hemi-protonated cytosine-cytosine+ (C:C+ ) base pairs. The growing interest in the i-DNA structure as a target in anticancer therapy increases the need for tools for a rapid and meaningful interpretation of the spectroscopic data of i-DNA samples. Herein, we analyzed the circular dichroism (CD) and thermal difference UV-absorbance spectra (TDS) of 255 DNA sequences by means of multivariate data analysis, aiming at unveiling peculiar spectral regions that could be used as diagnostic features during the analysis of i-DNA-forming sequences.

Thermal and pH Stabilities of i-DNA: Confronting in vitro Experiments with Models and In-Cell NMR Data.

Recent studies indicate that i-DNA, a four-stranded cytosine-rich DNA also known as the i-motif, is actually formed in vivo; however, a systematic study on sequence effects on stability has been missing. Herein, an unprecedented number of different sequences (271) bearing four runs of 3-6 cytosines with different spacer lengths has been tested. While i-DNA stability is nearly independent on total spacer length, the central spacer plays a special role on stability. Stability also depends on the length of the C-tracts at both acidic and neutral pHs. This study provides a global picture on i-DNA stability thanks to the large size of the introduced data set; it reveals unexpected features and allows to conclude that determinants of i-DNA stability do not mirror those of G-quadruplexes. Our results illustrate the structural roles of loops and C-tracts on i-DNA stability, confirm its formation in cells, and allow establishing rules to predict its stability.

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 Tm as high as 17°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.

Highly active G-quadruplex/hemin DNAzyme for sensitive colorimetric determination of lead(II).

A UV-vis, CD, and differential pulse voltammetric study was performed on the deactivation of the activity of parallel G-quadruplex/hemin DNAzymes (G4 DNAzymes) by Pb(II). The G4 DNAzyme carries a d[TC] sequence at its 3' end and is stabilized by potassium(I). On addition of Pb(II), the K(I) ions in the parallel G4 are replaced by Pb(II) to keep the parallel topology. Intruded Pb(II) decrease the affinity between the topology and hemin, this leads to a decrease of DNAzyme activity for catalyzing the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by hydrogen peroxide to form a green dye with an absorption maximum at 420 nm. The assay does not use any amplification, and has a linear response in the 0.01 to 10 µM Pb(II) concentration range and a 7.1 nM limit of detection. The method was successfully applied to the analysis of spiked water samples. Graphical abstractSchematic diagram of the colorimetric lead(II) assay based on the competition between K+ and Pb2+ stabilized G-quadruplex/hemin DNAzymes (G4 DNAzymes).

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 CuII 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.

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 100mM 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. 1D 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.

A Thermophilic Tetramolecular G-Quadruplex/Hemin DNAzyme.

The quadruplex-based DNAzyme system is one of the most useful artificial enzymes or catalysts; their unique properties make them reliable alternatives to proteins for performing catalytic transformation. The first prototype of a thermally stable DNAzyme system is presented. This thermophilic DNAzyme is capable of oxidizing substrates at high temperatures (up to 95 °C) and long reaction times (up to 18 h at 75 °C). The catalytic activity of the DNAzymes were investigated with the standard peroxidase-mimicking oxidation of 2,2'-azino-bis(3-ethylbenzothiozoline-6-sulfonic acid) (ABTS) by H2 O2 . The step-by-step design of this unique heat-activated G-quadruplex/hemin catalyst, including the modification of adenines at both ends of G-tracts, the choice of cation, and its concentration for DNAzyme stabilization, is described. This work investigates thoroughly the molecular basis of these catalytic properties and provides an example of an industrially relevant application.

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 (G4 ) units and exhibit structure-based stereocontrol for a series of reactions. However, for more biologically relevant higher-order HT G4 -DNAs (beyond a single G4 unit), the catalytic performances are unknown. Here, we found that higher-order HT G4 -DNA copper metalloenzymes (two or three G4 units) afford remarkably higher enantioselectivity (>90 % ee) and a five- to sixfold rate increase, compared to a single G4 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 G4 -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 G4 -DNA-based catalysis.

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

While 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 a multimerization effect applied to catalytic DNAs (or DNAzymes) and demonstrate that the enzymatic efficiency of G-quadruplexes (GQs) in interaction with the 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 providing new perspectives for biotechnological application of GQs.

An oxidatively damaged G-quadruplex/hemin DNAzyme.

Oxidative damage of guanine to 8-oxoguanine triggers a partial and variable loss of G-quadruplex/hemin DNAzyme activity and provides clues to the mechanistic origins of DNAzyme deactivation, which originates from an interplay between decreased G-quadruplex stability, lower hemin affinity and a modification of the nature of hemin binding sites.

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.

Enantioselective sulfoxidation reaction catalyzed by a G-quadruplex DNA metalloenzyme.

Enantioselective sulfoxidation reaction is achieved for the first time by a DNA metalloenzyme assembled with the human telomeric G-quadruplex DNA and Cu(ii)-4,4'-bimethyl-2,2'-bipyridine complex, and the mixed G-quadruplex architectures are responsible for the catalytic enantioselectivity and activity.