scholarly journals Investigation of ‘Head-to-Tail’-Connected Oligoaryl N,O-Ligands as Recognition Motifs for Cancer-Relevant G-Quadruplexes

Molecules ◽  
2017 ◽  
Vol 22 (12) ◽  
pp. 2160 ◽  
Author(s):  
Natalia Rizeq ◽  
Savvas Georgiades
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Biomolecular Recognition ◽  
Binding Affinities ◽  
Myc Oncogene ◽  
Recognition Element ◽  
G Quadruplex ◽  
Recognition Motifs ◽  
Fluorescence Titrations ◽  
Oligomeric Compounds

Oligomeric compounds, constituted of consecutive N,O-heteroaromatic rings, introduce useful and tunable properties as alternative ligands for biomolecular recognition. In this study, we have explored a synthetic scheme relying on Van Leusen oxazole formation, in conjunction with C–H activation of the formed oxazoles and their subsequent C–C cross-coupling to 2-bromopyridines in order to assemble a library of variable-length, ‘head-to-tail’-connected, pyridyl-oxazole ligands. Through investigation of the interaction of the three longer ligands (5-mer, 6-mer, 7-mer) with cancer-relevant G-quadruplex structures (human telomeric/22AG and c-Myc oncogene promoter/Myc2345-Pu22), the asymmetric pyridyl-oxazole motif has been demonstrated to be a prominent recognition element for G-quadruplexes. Fluorescence titrations reveal excellent binding affinities of the 7-mer and 6-mer for a Na+-induced antiparallel 22AG G-quadruplex (KD = 0.6 × 10−7 M−1 and 0.8 × 10−7 M−1, respectively), and satisfactory (albeit lower) affinities for the 22AG/K+ and Myc2345-Pu22/K+ G-quadruplexes. All ligands tested exhibit substantial selectivity for G-quadruplex versus duplex (ds26) DNA, as evidenced by competitive Förster resonance energy transfer (FRET) melting assays. Additionally, the 7-mer and 6-mer are capable of promoting a sharp morphology transition of 22AG/K+ G-quadruplex.

scholarly journals PhenQE8, a Novel Ligand of the Human Telomeric Quadruplex

2021 ◽  
Vol 22 (2) ◽  
pp. 749
Author(s):  
Patricia B. Gratal ◽  
Julia G. Quero ◽  
Adrián Pérez-Redondo ◽  
Zoila Gándara ◽  
Lourdes Gude
Keyword(s):  
Equilibrium Dialysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
The Novel ◽  
X Ray Diffraction ◽  
Telomeric Dna ◽  
Healthy Human ◽  
Quadruplex Dna ◽  
Step Procedure ◽  
G Quadruplex

A novel quadruplex ligand based on 1,10-phenanthroline and incorporating two guanyl hydrazone functionalities, PhenQE8, is reported herein. Synthetic access was gained in a two-step procedure with an overall yield of 61%. X-ray diffraction studies revealed that PhenQE8 can adopt an extended conformation that may be optimal to favor recognition of quadruplex DNA. DNA interactions with polymorphic G-quadruplex telomeric structures were studied by different techniques, such as Fluorescence resonance energy transfer (FRET) DNA melting assays, circular dichroism and equilibrium dialysis. Our results reveal that the novel ligand PhenQE8 can efficiently recognize the hybrid quadruplex structures of the human telomeric DNA, with high binding affinity and quadruplex/duplex selectivity. Moreover, the compound shows significant cytotoxic activity against a selected panel of cultured tumor cells (PC-3, HeLa and MCF-7), whereas its cytotoxicity is considerably lower in healthy human cells (HFF-1 and RPWE-1).

scholarly journals G-Quadruplexes and Their Ligands: Biophysical Methods to Unravel G-Quadruplex/Ligand Interactions

2021 ◽  
Vol 14 (8) ◽  
pp. 769
Author(s):  
Tiago Santos ◽  
Gilmar F. Salgado ◽  
Eurico J. Cabrita ◽  
Carla Cruz
Keyword(s):  
High Throughput ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Mode ◽  
Titration Calorimetry ◽  
Apparent Affinity ◽  
G Quadruplex ◽  
Ligand Interactions ◽  
Types Of Information ◽  
Fluorescent Intercalator Displacement

Progress in the design of G-quadruplex (G4) binding ligands relies on the availability of approaches that assess the binding mode and nature of the interactions between G4 forming sequences and their putative ligands. The experimental approaches used to characterize G4/ligand interactions can be categorized into structure-based methods (circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography), affinity and apparent affinity-based methods (surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and mass spectrometry (MS)), and high-throughput methods (fluorescence resonance energy transfer (FRET)-melting, G4-fluorescent intercalator displacement assay (G4-FID), affinity chromatography and microarrays. Each method has unique advantages and drawbacks, which makes it essential to select the ideal strategies for the biological question being addressed. The structural- and affinity and apparent affinity-based methods are in several cases complex and/or time-consuming and can be combined with fast and cheap high-throughput approaches to improve the design and development of new potential G4 ligands. In recent years, the joint use of these techniques permitted the discovery of a huge number of G4 ligands investigated for diagnostic and therapeutic purposes. Overall, this review article highlights in detail the most commonly used approaches to characterize the G4/ligand interactions, as well as the applications and types of information that can be obtained from the use of each technique.

scholarly journals A tunable assay for modulators of genome-destabilizing DNA structures

2019 ◽  
Vol 47 (13) ◽  
pp. e73-e73 ◽  
Author(s):  
Imee M A del Mundo ◽  
Eun Jeong Cho ◽  
Kevin N Dalby ◽  
Karen M Vasquez
Keyword(s):  
Dna Sequences ◽  
Genetic Instability ◽  
Human Cancer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dna Structures ◽  
Myc Oncogene ◽  
Human Genomes ◽  
Complementary Oligonucleotide ◽  
Translocation Breakpoints

Abstract Regions of genomic instability are not random and often co-localize with DNA sequences that can adopt alternative DNA structures (i.e. non-B DNA, such as H-DNA). Non-B DNA-forming sequences are highly enriched at translocation breakpoints in human cancer genomes, representing an endogenous source of genetic instability. However, a further understanding of the mechanisms involved in non-B DNA-induced genetic instability is needed. Small molecules that can modulate the formation/stability of non-B DNA structures, and therefore the subsequent mutagenic outcome, represent valuable tools to study DNA structure-induced genetic instability. To this end, we have developed a tunable Förster resonance energy transfer (FRET)-based assay to detect triplex/H-DNA-destabilizing and -stabilizing ligands. The assay was designed by incorporating a fluorophore-quencher pair in a naturally-occurring H-DNA-forming sequence from a chromosomal breakpoint hotspot in the human c-MYC oncogene. By tuning triplex stability via buffer composition, the assay functions as a dual-reporter that can identify stabilizers and destabilizers, simultaneously. The assay principle was demonstrated using known triplex stabilizers, BePI and coralyne, and a complementary oligonucleotide to mimic a destabilizer, MCRa2. The potential of the assay was validated in a 384-well plate with 320 custom-assembled compounds. The discovery of novel triplex stabilizers/destabilizers may allow the regulation of genetic instability in human genomes.

scholarly journals A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

2019 ◽  
Author(s):  
Chandani Warnasooriya ◽  
Eliezra Glasser ◽  
Callen F. Feeney ◽  
Kholiswa M. Laird ◽  
Jermaine L. Jenkins ◽  
...  
Keyword(s):  
Single Molecule ◽  
Splice Site ◽  
Conformational Dynamics ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Splicing Factors ◽  
Rna Recognition ◽  
Rna Recognition Motifs ◽  
Recognition Motifs ◽  
Rna Complexes

ABSTRACTAn essential heterodimer of the U2AF1 and U2AF2 pre-mRNA splicing factors nucleates spliceosome assembly at polypyrimidine (Py) signals preceding the major class of 3ʹ splice sites. Among myelodysplastic syndromes (MDS), U2AF1 frequently acquires an S34F-encoding mutation. The influence of the U2AF1 subunit and its S34F mutation on the U2AF2 conformations remains unknown. Here, we employ single molecule Förster resonance energy transfer (FRET) to determine the influence of wild-type or S34F-substituted U2AF1 on the conformational dynamics of U2AF2 and its splice site RNA complexes. In the absence of RNA, the U2AF1 subunit stabilizes a high FRET value, which by structure-guided mutagenesis corresponds to a closed conformation of the tandem U2AF2 RNA recognition motifs (RRMs). When the U2AF heterodimer is bound to a strong, uridine-rich splice site, U2AF2 switches to a lower FRET value characteristic of an open, side-by-side arrangement of the RRMs. Remarkably, the U2AF heterodimer binds weak, uridine-poor Py tracts as a mixture of closed and open U2AF2 conformations, which are modulated by the S34F mutation. Shifts between open and closed U2AF2 may underlie U2AF1-dependent splicing of degenerate Py tracts and contribute to a subset of S34F-dysregulated splicing events in MDS patients.

scholarly journals A mechanism for the extension and unfolding of parallel telomeric G-quadruplexes by human telomerase at single-molecule resolution

2020 ◽  
Author(s):  
Bishnu P. Paudel ◽  
Aaron Lavel Moye ◽  
Hala Abou Assi ◽  
Roberto El-Khoury ◽  
Scott B. Cohen ◽  
...  
Keyword(s):  
Single Molecule ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Quadruplex Structure ◽  
G Quadruplex ◽  
Nucleotide Addition ◽  
Small Molecule Ligands ◽  
Large Conformational Change ◽  
Human Telomerase ◽  
Linear Product

AbstractTelomeric G-quadruplexes (G4) were long believed to form a protective structure at telomeres, preventing their extension by the ribonucleoprotein telomerase. Contrary to this belief, we have previously demonstrated that parallel-stranded conformations of telomeric G4 can be extended by human and ciliate telomerase. However, a mechanistic understanding of the interaction of telomerase with structured DNA remained elusive. Here, we use single-molecule fluorescence resonance energy transfer (smFRET) microscopy and bulk-phase enzymology to propose a mechanism for the resolution and extension of parallel G4 by telomerase. Binding is initiated by the RNA template of telomerase interacting with the G-quadruplex; nucleotide addition then proceeds to the end of the RNA template. It is only through the large conformational change of translocation following synthesis that the G-quadruplex structure is completely unfolded to a linear product. Surprisingly, parallel G4 stabilization with either small molecule ligands or by chemical modification does not always inhibit G4 unfolding and extension by telomerase. These data reveal that telomerase is a parallel G-quadruplex resolvase.

Fluorescence resonance energy transfer-based DNA framework assembled split G-quadruplex nanodevices for microRNA sensing

2020 ◽  
Vol 56 (88) ◽  
pp. 13583-13586
Author(s):  
Gaoxing Su ◽  
Min Zhu ◽  
Mengting Xu ◽  
Jianbin Pan ◽  
Yao Zhou ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
G Quadruplex ◽  
Fluorescence Resonance

DNA framework assembled split G4 nanodevice was fabricated to realize the microRNA imaging in living cells.

scholarly journals Interactions Between Spermine-Derivatized Tentacle Porphyrins and The Human Telomeric DNA G-Quadruplex

2018 ◽  
Vol 19 (11) ◽  
pp. 3686 ◽  
Author(s):  
Navin Sabharwal ◽  
Jessica Chen ◽  
Joo Lee ◽  
Chiara Gangemi ◽  
Alessandro D'Urso ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Binding Constants ◽  
Spectroscopic Studies ◽  
Resonance Light Scattering ◽  
Binding Modes ◽  
Telomeric Dna ◽  
Binding Stoichiometry ◽  
G Quadruplex

G-rich DNA sequences have the potential to fold into non-canonical G-Quadruplex (GQ) structures implicated in aging and human diseases, notably cancers. Because stabilization of GQs at telomeres and oncogene promoters may prevent cancer, there is an interest in developing small molecules that selectively target GQs. Herein, we investigate the interactions of meso-tetrakis-(4-carboxysperminephenyl)porphyrin (TCPPSpm4) and its Zn(II) derivative (ZnTCPPSpm4) with human telomeric DNA (Tel22) via UV-Vis, circular dichroism (CD), and fluorescence spectroscopies, resonance light scattering (RLS), and fluorescence resonance energy transfer (FRET) assays. UV-Vis titrations reveal binding constants of 4.7 × 106 and 1.4 × 107 M−1 and binding stoichiometry of 2–4:1 and 10–12:1 for TCPPSpm4 and ZnTCPPSpm4, respectively. High stoichiometry is supported by the Job plot data, CD titrations, and RLS data. FRET melting indicates that TCPPSpm4 stabilizes Tel22 by 36 ± 2 °C at 7.5 eq., and that ZnTCPPSpm4 stabilizes Tel22 by 33 ± 2 °C at ~20 eq.; at least 8 eq. of ZnTCPPSpm4 are required to achieve significant stabilization of Tel22, in agreement with its high binding stoichiometry. FRET competition studies show that both porphyrins are mildly selective for human telomeric GQ vs duplex DNA. Spectroscopic studies, combined, point to end-stacking and porphyrin self-association as major binding modes. This work advances our understanding of ligand interactions with GQ DNA.

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