Oscar Mendoza

G-quadruplexes and helicases

Guanine-rich DNA strands can fold in vitro into non-canonical DNA structures called G-quadruplexes. These structures may be very stable under physiological conditions. Evidence suggests that G-quadruplex structures may act as ‘knots’ within genomic DNA, and it has been hypothesized that proteins may have evolved to remove these structures. The first indication of how G-quadruplex structures could be unfolded enzymatically came in the late 1990s with reports that some well-known duplex DNA helicases resolved these structures in vitro. Since then, the number of studies reporting G-quadruplex DNA unfolding by helicase enzymes has rapidly increased. The present review aims to present a general overview of the helicase/G-quadruplex field.

Assembly of Palladium(II) and Platinum(II) Metallo‐Rectangles with a Guanosine‐Substituted Terpyridine and Study of Their Interactions with Quadruplex DNA

Two novel [2+2] metallo-assemblies based on a guanosine-substituted terpyridine ligand (1) coordinated to palladium(II) (2 a) and platinum(II) (2 b) are reported. These supramolecular assemblies have been fully characterized by NMR spectroscopy, ESI mass spectrometry and elemental analyses. The palladium(II) complex (2 a) has also been characterized by single crystal X-ray diffraction studies confirming that the system is a [2+2] metallo-rectangle in the solid state. The stabilities of these [2+2] assemblies in solution have been confirmed by DOSY studies as well as by variable temperature (1)H NMR spectroscopy. The ability of these dinuclear complexes to interact with quadruplex and duplex DNA was investigated by fluorescent intercalator displacement (FID) assays, fluorescence resonance energy transfer (FRET) melting studies, and electrospray mass spectrometry (ESI-MS). These studies have shown that both these assemblies interact selectively with quadruplex DNA (human telomeric DNA and the G-rich promoter region of c-myc oncogene) over duplex DNA, and are able to induce dimerization of parallel G-quadruplex structures.

Salphen metal complexes as tunable G-quadruplex binders and optical probes

A series of seven new metal complexes (metal = NiII, CuII, PtII and VO2+) with substituted salphen ligands have been prepared and their duplex and G-quadruplex DNA affinities determined. The selectivity of the complexes towards a given DNA topology is dictated by several factors including geometry, overall charge and substitution pattern of the complex. We also show that the two platinum(II)–salphen complexes developed as part of this series are emissive. Confocal microscopy studies were carried out with these two complexes using four different cell lines (CHO, HeLa, U2OS and HepG2). These studies showed that the cell permeability and localization are different for the two probes and highly dependent on the cell line used.

A fluorescence-based helicase assay: application to the screening of G-quadruplex ligands

Helicases, enzymes that unwind DNA or RNA structure, are present in the cell nucleus and in the mitochondrion. Although the majority of the helicases unwind DNA or RNA duplexes, some of these proteins are known to resolve unusual structures such as G-quadruplexes (G4) in vitro. G4 may form stable barrier to the progression of molecular motors tracking on DNA. Monitoring G4 unwinding by these enzymes may reveal the mechanisms of the enzymes and provides information about the stability of these structures. In the experiments presented herein, we developed a reliable, inexpensive and rapid fluorescence-based technique to monitor the activity of G4 helicases in real time in a 96-well plate format. This system was used to screen a series of G4 structures and G4 binders for their effect on the Pif1 enzyme, a 5′ to 3′ DNA helicase. This simple assay should be adaptable to analysis of other helicases and G4 structures.

Quadruplex DNA-Stabilising Dinuclear Platinum(II) Terpyridine Complexes with Flexible Linkers.

Four dinuclear terpyridineplatinum(II) (Pt-terpy) complexes were investigated for interactions with G-quadruplex DNA (QDNA) and duplex DNA (dsDNA) by synchrotron radiation circular dichroism (SRCD), fluorescent intercalator displacement (FID) assays and fluorescence resonance energy transfer (FRET) melting studies. Additionally, computational docking studies were undertaken to provide insight into potential binding modes for these complexes. The complexes demonstrated the ability to increase the melting temperature of various QDNA motifs by up to 17 °C and maintain this in up to a 600-fold excess of dsDNA. This study demonstrates that dinuclear Pt-terpy complexes stabilise QDNA and have a high degree of selectivity for QDNA over dsDNA.

5' to 3' Unfolding Directionality of DNA Secondary Structures by Replication Protein A: G-QUADRUPLEXES AND DUPLEXES.

The replication protein A (RPA) is a single-stranded DNA-binding protein that plays an essential role in DNA metabolism. RPA is able to unfold G-quadruplex (G4) structures formed by telomeric DNA sequences, a function important for telomere maintenance. To elucidate the mechanism through which RPA unfolds telomeric G4s, we studied its interaction with oligonucleotides that adopt a G4 structure extended with a single-stranded tail on either side of the G4. Binding and unfolding was characterized using several biochemical and biophysical approaches and in the presence of specific G4 ligands, such as telomestatin and 360A. Our data show that RPA can bind on each side of the G4 but it unwinds the G4 only from 5′ toward 3′. We explain the 5′ to 3′ unfolding directionality in terms of the 5′ to 3′ oriented laying out of hRPA subunits along single-stranded DNA. Furthermore, we demonstrate by kinetics experiments that RPA proceeds with the same directionality for duplex unfolding.

Orienting Tetramolecular G‐Quadruplex Formation: The Quest for the Elusive RNA Antiparallel Quadruplex

DNA and RNA G-quadruplexes (G4) are unusual nucleic acid structures involved in a number of key biological processes. RNA G-quadruplexes are less studied although recent evidence demonstrates that they are biologically relevant. Compared to DNA quadruplexes, RNA G4 are generally more stable and less polymorphic. Duplexes and quadruplexes may be combined to obtain pure tetrameric species. Here, we investigated whether classical antiparallel duplexes can drive the formation of antiparallel tetramolecular quadruplexes. This concept was first successfully applied to DNA G4. In contrast, RNA G4 were found to be much more unwilling to adopt the forced antiparallel orientation, highlighting that the reason RNA adopts a different structure must not be sought in the loops but in the G-stem structure itself. RNA antiparallel G4 formation is likely to be restricted to a very small set of peculiar sequences, in which other structural features overcome the formidable intrinsic barrier preventing its formation.

Design, Synthesis, and Evaluation of 2,9-Bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline Derivatives as G-Quadruplex Ligands.

Genomic sequences able to form guanine quadruplexes (G4) are found in oncogene promoters, in telomeres, and in 5′‐ and 3′‐untranslated regions as well as introns of messenger RNAs. These regions are potential targets for drugs designed to treat cancer. Herein, we present the design and syntheses of ten new phenanthroline derivatives and characterization of their interactions with G4‐forming oligonucleotides. We evaluated ligand‐induced stabilization and specificity and selectivity of ligands for various G4 conformations using FRET‐melting experiments. We investigated the interaction of compound 1 a (2,9‐bis{4‐[(3‐dimethylaminopropyl)aminomethyl]phenyl}‐1,10‐phenanthroline), which combined the greatest stabilizing effect and specificity for G4, with human telomeric sequences using FRET, circular dichroism, and ESI‐MS. In addition, we showed that compound 1 a interferes with the G4 helicase activity of Saccharomyces cerevisiae Pif1. Interestingly, compound 1 a was significantly more cytotoxic toward two human leukemic cell lines than to normal human blood mononuclear cells. These novel phenanthroline derivatives will be a starting point for further development and optimization of potent G4 ligands that have potential as anticancer agents.

Spectroscopic data for the G-quadruplex DNA to duplex DNA reaction.

This article describes additional data related to a research article entitled “Kinetics of Quadruplex to Duplex Conversion” (Mendoza et al. 2015 [1]). We followed the opening reaction of a series of intramolecular G-quadruplex structures by the addition of their corresponding complementary strand. Fluorolabeled complementary strands allowed to monitor the reaction in real-time. An adapted kinetic model was then applied in order to obtain the kinetic parameters of this reaction. We present a series of kinetic traces providing raw data of the G4 opening reaction and the fitting model applied in every case. In addition CD spectra and UV melting data is also provided to confirm the stability of all the DNA structures considered (G-quadruplex and duplex DNA).

DNA Nanodevices Confined on DNA Origamis: an Isothermal Ratchet Driven by Boundary Conditions

In this note we investigate the diffusive behaviour and the boundary conditions of DNA nanodevices using the toe hold mediated strand displacement method. The goal is to extract the basic principles governing the difference observed in diluted solution and in confined environment where the devices are tethered on a DNA Origami. We note that the excluded volume interaction between the two strands running in opposite direction must give a sub diffusive behaviour that can lead to very long waiting times between jumps. When the Boundary Conditions generate a strong asymmetry in the device, the probability to perform the logical operation, thus to remove the output strand, is one. However, we envision unexpected marked differences between diluted solutions and confined environment both for controlling the boundary condition and the sub diffusive behaviour. These differences rise new questions on the interest to use the toe hold mediated strand displacement method on DNA Origamis.