Rudimentary G-Quadruplex-Based Telomere Capping In Saccharomyces Cerevisiae

Telomere capping conceals chromosome ends from exonucleases and checkpoints, but the full range of capping mechanisms is not well defined. Telomeres have the potential to form G-quadruplex (G4) DNA, although evidence for telomere G4 DNA function in vivo is limited. In budding yeast, capping requires the Cdc13 protein and is lost at nonpermissive temperatures in cdc13-1 mutants. Here, we use several independent G4 DNA-stabilizing treatments to suppress cdc13-1 capping defects. These include overexpression of three different G4 DNA binding proteins, loss of the G4 DNA unwinding helicase Sgs1, or treatment with small molecule G4 DNA ligands. In vitro, we show that protein-bound G4 DNA at a 3\u27 overhang inhibits 5\u27-\u3e 3\u27 resection of a paired strand by exonuclease I. These findings demonstrate that, at least in the absence of full natural capping, G4 DNA can play a positive role at telomeres in vivo

Improved performances of catalytic G-quadruplexes (G4-DNAzymes) via the chemical modifications of the DNA backbone to provide Gquadruplexes with double 3′-external G-quartets

Here we report on the design of a new catalytic G-quadruplex-DNA system (G4-DNAzyme) based on the modification of the DNA scaffold to provide the DNA pre-catalyst with two identical 3′-ends, known to bemore catalytically proficient than the 5′-ends. To this end, we introduced a 5′-5′ inversion of polarity site in the middle of the G4-forming sequences AG4A andAG6A to obtain d(3′AGG5′-5′GGA3′) (orAG2-G2A) and d(3′AGGG5′-5′GGGA3′) (or AG3-G3A) that fold into stable G4 whose tetramolecular nature was confirmed via nuclear magnetic resonance (NMR) and circular dichroism(CD) investigations. Both AG2-G2AandAG3-G3A display two identical external G-quartets (3′-ends) known to interact with the cofactor hemin with a high efficiency, making the resulting complex competent to performhemoprotein-like catalysis (G4-DNAzyme). A systematic comparison of the performances of modified and unmodified G4s lends credence to the relevance of the modification exploited here (5′-5′ inversion of polarity site), which represents a new chemical opportunity to improve the overall activity of catalytic G4s

“One Ring to Bind Them All”—Part I: The Efficiency of the Macrocyclic Scaffold for G-Quadruplex DNA Recognition

Macrocyclic scaffolds are particularly attractive for designing selective G-quadruplex ligands essentially because, on one hand, they show a poor affinity for the “standard” B-DNA conformation and, on the other hand, they fit nicely with the external G-quartets of quadruplexes. Stimulated by the pioneering studies on the cationic porphyrin TMPyP4 and the natural product telomestatin, follow-up studies have developed, rapidly leading to a large diversity of macrocyclic structures with remarkable-quadruplex binding properties and biological activities. In this review we summarize the current state of the art in detailing the three main categories of quadruplex-binding macrocycles described so far (telomestatin-like polyheteroarenes, porphyrins and derivatives, polyammonium cyclophanes), and in addressing both synthetic issues and biological aspects

“One Ring to Bind Them All”—Part II: Identification of Promising G-Quadruplex Ligands by Screening of Cyclophane-Type Macrocycles

A collection of 26 polyammonium cyclophane-type macrocycles with a large structural diversity has been screened for G-quadruplex recognition. A two-step selection procedure based on the FRET-melting assay was carried out enabling identification of macrocycles of high affinity (ΔT1/2 up to 30°C) and high selectivity for the human telomeric G-quadruplex. The four selected hits possess sophisticated architectures, more particularly the presence of a pendant side-arm as well as the existence of a particular topological arrangement appear to be strong determinants of quadruplex binding. These compounds are thus likely to create multiple contacts with the target that may be at the origin of their high selectivity, thereby suggesting that this class of macrocycles offers unique advantages for targeting G-quadruplex-DNA

Cellular modulation of a G-quadruplex structure found in the lung cancer-related microRNA-3196

Funding Information: Daniela Alexandre and André Miranda acknowledge the doctoral fellowship grants from FCT – Foundation for Science and Technology ref. 2021.07695.BD and 2021.04785.BD, respectively. Thanks are due to CICS-UBI program funding DOI 10.54499/UIDB/00709/2020 (https://doi.org/10.54499/UIDB/00709/2020) and the CICS-UBI program funding with DOI 10.54499/UIDP/00709/2020 (https://doi.org/10.54499/UIDP/00709/2020) with national funds from the Foundation for Science and Technology, PPBI-Portuguese Platform of BioImaging research unit (POCI-01-0145-FEDER-022122), and to the Portuguese NMR Network (ROTEIRO/0031/2013-PINFRA/22161/2016), through national funds and, where applicable, co-financed by the FEDER through COMPETE 2020, POCI, PORL and PIDDAC. C.C. acknowledges the grants from project PAPILOMA ref. CENTRO-01-0145-FEDER-181235, NRC-LPCC Bolsa Dr. Rocha Alves 2022, Instruct-ERIC Pilot R&D application ID 2473. This work was supported by EATRIS, the European infrastructure for translational medicine. Publisher Copyright: © 2025 The AuthorsRNA G-quadruplexes (G4s) are promising drug targets due to their high cellular abundance. G-rich RNA regions inherently form G4 structures, while GC-rich sequences adopt stem-loop conformations, and their dynamic equilibrium critically influences RNA function. MicroRNAs (miRs), key regulators of protein expression, undergo processing by Dicer, which specifically recognizes stem-loop structures in precursor miRs (pre-miRs). Notably, some pre-miRs containing G4-forming sequences influence Dicer cleavage, suggesting that G4s can directly regulate miR production. Moreover, pre-miRs with G4 structures present promising targets for small molecules. This research focuses on identifying and modulating G4 structure in pre-miR-3196 to restore normal lung cancer (LC) levels, offering a potential therapeutic strategy. Firstly, bioinformatic analyses indicated the presence of G4 motifs in pre-miR-3196. We then demonstrated in vitro that this RNA sequence folds into stable G4s by a combination of biophysical and biochemical assays. Then, we demonstrated the formation of these G4s in human cancer cells by confocal imaging before showing that these G4s can be modulated using the RNA G4 destabilizer PhpC, which impacts the miR-3196 biogenesis. These findings highlighted the possibility of using G4s to control the expression of mature miR-3196 and revealed the potential of using the destabilizer PhpC to adjust its G4 structure.publishersversionpublishe

PIRH2-Dependent Dna Damage in Neurons Induced by the G-Quadruplex Ligand Pyridostatin

Noncanonical base pairing between four guanines (G) within single-stranded G-rich sequences leads to formation of а G-quartet. Self-stacking of G-quartets results in a columnar four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA regulates multiple DNA-dependent processes, including transcription, replication, and telomere function. How G4s function in neurons is poorly understood. Here, we performed a genome-wide gene expression analysis (RNA-Seq) to identify genes modulated by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS promotes stabilization of G4 structures, thus allowing us to define genes directly or indirectly responsive to G4 regulation. We found that 901 genes were differentially expressed in neurons treated with PDS out of a total of 18,745 genes with measured expression. Of these, 505 genes were downregulated and 396 genes were upregulated and included gene networks regulating p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), a modulator of DNA damage responses, was upregulated by PDS. Ectopically overexpressing Pirh2 promoted the formation of DNA double-strand breaks, suggesting a new DNA damage mechanism in neurons that is regulated by G4 stabilization. Pirh2 downregulated DDX21, an RNA helicase that unfolds G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the neuronal nucleolus. Our data reveal the genes that are responsive to PDS treatment and suggest similar transcriptional regulation by endogenous G4-DNA ligands. They also connect G4-dependent regulation of transcription and DNA damage mechanisms in neuronal cells

Small-molecule G-quadruplex stabilizers reveal a novel pathway of autophagy regulation in neurons

International audienc

Computational understanding and experimental characterization of twice-as-smart quadruplex ligands as chemical sensors of bacterial nucleotide second messengers

A twice-as-smart ligand is a small molecule that experiences a structural switch upon interaction with its target (i.e., smart ligand) that concomitantly triggers its fluorescence(i.e., smart probe). Prototypes of twice-as-smart ligands were recently developed to track and label G-quadruplexes: these higher-order nucleic acid structures originate in the assembly of four guanine(G)-rich DNA or RNA strands, whose stability is imparted by the formation and the self-assembly of G-quartets. The firstprototypes of twice-as-smart quadruplex ligands were designed to exploit the self-association of quartets, being themselves synthetic G-quartets. While their quadruplex recognition capability has been thoroughly documented, some doubts remain about the precise photophysical mechanism that underlies their peculiar spectroscopic properties. Here, we uncovered this mechanism via complete theoretical calculations. Collected information was then used to develop of a novel application of twice-as-smart ligands, as efficnt chemical sensors of bacterial signaling pathways via the fluorescentdetection of naturally occurring extracellular quadruplexes formed by cyclic dimeric guanosine monophosphate (c-di-GMP)

Why does the pUG tail curl?

International audienceRoschdi et al. report on a new higher-order RNA structure folding from an alternating uridine (U)/guanosine (G) repeated sequence-the pUG tail-into a peculiar G-quadruplex structurethe pUG fold-found to orchestrate the gene-silencing activity of pUGylated RNAs