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1
Desai, Nakshi, Viraj Shah, and Bhaskar Datta. "Assessing G4-Binding Ligands In Vitro and in Cellulo Using Dimeric Carbocyanine Dye Displacement Assay." Molecules 26, no. 5 (March 5, 2021): 1400. http://dx.doi.org/10.3390/molecules26051400.
Повний текст джерелаАнотація:
G-quadruplexes (G4) are the most actively studied non-canonical secondary structures formed by contiguous repeats of guanines in DNA or RNA strands. Small molecule mediated targeting of G-quadruplexes has emerged as an attractive tool for visualization and stabilization of these structures inside the cell. Limited number of DNA and RNA G4-selective assays have been reported for primary ligand screening. A combination of fluorescence spectroscopy, AFM, CD, PAGE, and confocal microscopy have been used to assess a dimeric carbocyanine dye B6,5 for screening G4-binding ligands in vitro and in cellulo. The dye B6,5 interacts with physiologically relevant DNA and RNA G4 structures, resulting in fluorescence enhancement of the molecule as an in vitro readout for G4 selectivity. Interaction of the dye with G4 is accompanied by quadruplex stabilization that extends its use in primary screening of G4 specific ligands. The molecule is cell permeable and enables visualization of quadruplex dominated cellular regions of nucleoli using confocal microscopy. The dye is displaced by quarfloxin in live cells. The dye B6,5 shows remarkable duplex to quadruplex selectivity in vitro along with ligand-like stabilization of DNA G4 structures. Cell permeability and response to RNA G4 structures project the dye with interesting theranostic potential. Our results validate that B6,5 can serve the dual purpose of visualization of DNA and RNA G4 structures and screening of G4 specific ligands, and adds to the limited number of probes with such potential.
2
Zheng, Alice J.-L., Aikaterini Thermou, Pedro Guixens Gallardo, Laurence Malbert-Colas, Chrysoula Daskalogianni, Nathan Vaudiau, Petter Brohagen, et al. "The different activities of RNA G-quadruplex structures are controlled by flanking sequences." Life Science Alliance 5, no. 2 (November 16, 2021): e202101232. http://dx.doi.org/10.26508/lsa.202101232.
Повний текст джерелаАнотація:
The role of G-quadruplex (G4) RNA structures is multifaceted and controversial. Here, we have used as a model the EBV-encoded EBNA1 and the Kaposi’s sarcoma-associated herpesvirus (KSHV)-encoded LANA1 mRNAs. We have compared the G4s in these two messages in terms of nucleolin binding, nuclear mRNA retention, and mRNA translation inhibition and their effects on immune evasion. The G4s in the EBNA1 message are clustered in one repeat sequence and the G4 ligand PhenDH2 prevents all G4-associated activities. The RNA G4s in the LANA1 message take part in similar multiple mRNA functions but are spread throughout the message. The different G4 activities depend on flanking coding and non-coding sequences and, interestingly, can be separated individually. Together, the results illustrate the multifunctional, dynamic and context-dependent nature of G4 RNAs and highlight the possibility to develop ligands targeting specific RNA G4 functions. The data also suggest a common multifunctional repertoire of viral G4 RNA activities for immune evasion.
3
Asamitsu, Sefan, Masayuki Takeuchi, Susumu Ikenoshita, Yoshiki Imai, Hirohito Kashiwagi, and Norifumi Shioda. "Perspectives for Applying G-Quadruplex Structures in Neurobiology and Neuropharmacology." International Journal of Molecular Sciences 20, no. 12 (June 13, 2019): 2884. http://dx.doi.org/10.3390/ijms20122884.
Повний текст джерелаАнотація:
The most common form of DNA is a right-handed helix or the B-form DNA. DNA can also adopt a variety of alternative conformations, non-B-form DNA secondary structures, including the DNA G-quadruplex (DNA-G4). Furthermore, besides stem-loops that yield A-form double-stranded RNA, non-canonical RNA G-quadruplex (RNA-G4) secondary structures are also observed. Recent bioinformatics analysis of the whole-genome and transcriptome obtained using G-quadruplex–specific antibodies and ligands, revealed genomic positions of G-quadruplexes. In addition, accumulating evidence pointed to the existence of these structures under physiologically- and pathologically-relevant conditions, with functional roles in vivo. In this review, we focused on DNA-G4 and RNA-G4, which may have important roles in neuronal function, and reveal mechanisms underlying neurological disorders related to synaptic dysfunction. In addition, we mention the potential of G-quadruplexes as therapeutic targets for neurological diseases.
4
Yan, Kevin Kok-Phen, Ikenna Obi, and Nasim Sabouri. "The RGG domain in the C-terminus of the DEAD box helicases Dbp2 and Ded1 is necessary for G-quadruplex destabilization." Nucleic Acids Research 49, no. 14 (July 24, 2021): 8339–54. http://dx.doi.org/10.1093/nar/gkab620.
Повний текст джерелаАнотація:
Abstract The identification of G-quadruplex (G4) binding proteins and insights into their mechanism of action are important for understanding the regulatory functions of G4 structures. Here, we performed an unbiased affinity-purification assay coupled with mass spectrometry and identified 30 putative G4 binding proteins from the fission yeast Schizosaccharomyces pombe. Gene ontology analysis of the molecular functions enriched in this pull-down assay included mRNA binding, RNA helicase activity, and translation regulator activity. We focused this study on three of the identified proteins that possessed putative arginine-glycine-glycine (RGG) domains, namely the Stm1 homolog Oga1 and the DEAD box RNA helicases Dbp2 and Ded1. We found that Oga1, Dbp2, and Ded1 bound to both DNA and RNA G4s in vitro. Both Dbp2 and Ded1 bound to G4 structures through the RGG domain located in the C-terminal region of the helicases, and point mutations in this domain weakened the G4 binding properties of the helicases. Dbp2 and Ded1 destabilized less thermostable G4 RNA and DNA structures, and this ability was independent of ATP but dependent on the RGG domain. Our study provides the first evidence that the RGG motifs in DEAD box helicases are necessary for both G4 binding and G4 destabilization.
5
Wang, Shao-Ru, Yuan-Qin Min, Jia-Qi Wang, Chao-Xing Liu, Bo-Shi Fu, Fan Wu, Ling-Yu Wu, et al. "A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target." Science Advances 2, no. 4 (April 2016): e1501535. http://dx.doi.org/10.1126/sciadv.1501535.
Повний текст джерелаАнотація:
G-quadruplex (G4) is one of the most important secondary structures in nucleic acids. Until recently, G4 RNAs have not been reported in any ribovirus, such as the hepatitis C virus. Our bioinformatics analysis reveals highly conserved guanine-rich consensus sequences within the core gene of hepatitis C despite the high genetic variability of this ribovirus; we further show using various methods that such consensus sequences can fold into unimolecular G4 RNA structures, both in vitro and under physiological conditions. Furthermore, we provide direct evidences that small molecules specifically targeting G4 can stabilize this structure to reduce RNA replication and inhibit protein translation of intracellular hepatitis C. Ultimately, the stabilization of G4 RNA in the genome of hepatitis C represents a promising new strategy for anti–hepatitis C drug development.
6
Götz, Silvia, Satyaprakash Pandey, Sabrina Bartsch, Stefan Juranek, and Katrin Paeschke. "A Novel G-Quadruplex Binding Protein in Yeast—Slx9." Molecules 24, no. 9 (May 7, 2019): 1774. http://dx.doi.org/10.3390/molecules24091774.
Повний текст джерелаАнотація:
G-quadruplex (G4) structures are highly stable four-stranded DNA and RNA secondary structures held together by non-canonical guanine base pairs. G4 sequence motifs are enriched at specific sites in eukaryotic genomes, suggesting regulatory functions of G4 structures during different biological processes. Considering the high thermodynamic stability of G4 structures, various proteins are necessary for G4 structure formation and unwinding. In a yeast one-hybrid screen, we identified Slx9 as a novel G4-binding protein. We confirmed that Slx9 binds to G4 DNA structures in vitro. Despite these findings, Slx9 binds only insignificantly to G-rich/G4 regions in Saccharomyces cerevisiae as demonstrated by genome-wide ChIP-seq analysis. However, Slx9 binding to G4s is significantly increased in the absence of Sgs1, a RecQ helicase that regulates G4 structures. Different genetic and molecular analyses allowed us to propose a model in which Slx9 recognizes and protects stabilized G4 structures in vivo.
7
Illodo, Sara, Cibrán Pérez-González, Ramiro Barcia, Flor Rodríguez-Prieto, Wajih Al-Soufi, and Mercedes Novo. "Spectroscopic Characterization of Mitochondrial G-Quadruplexes." International Journal of Molecular Sciences 23, no. 2 (January 15, 2022): 925. http://dx.doi.org/10.3390/ijms23020925.
Повний текст джерелаАнотація:
Guanine quadruplexes (G4s) are highly polymorphic four-stranded structures formed within guanine-rich DNA and RNA sequences that play a crucial role in biological processes. The recent discovery of the first G4 structures within mitochondrial DNA has led to a small revolution in the field. In particular, the G-rich conserved sequence block II (CSB II) can form different types of G4s that are thought to play a crucial role in replication. In this study, we decipher the most relevant G4 structures that can be formed within CSB II: RNA G4 at the RNA transcript, DNA G4 within the non-transcribed strand and DNA:RNA hybrid between the RNA transcript and the non-transcribed strand. We show that the more abundant, but unexplored, G6AG7 (37%) and G6AG8 (35%) sequences in CSB II yield more stable G4s than the less profuse G5AG7 sequence. Moreover, the existence of a guanine located 1 bp upstream promotes G4 formation. In all cases, parallel G4s are formed, but their topology changes from a less ordered to a highly ordered G4 when adding small amounts of potassium or sodium cations. Circular dichroism was used due to discriminate different conformations and topologies of nucleic acids and was complemented with gel electrophoresis and fluorescence spectroscopy studies.
8
Falabella, Micol, Rafael J. Fernandez, F. Brad Johnson, and Brett A. Kaufman. "Potential Roles for G-Quadruplexes in Mitochondria." Current Medicinal Chemistry 26, no. 16 (August 26, 2019): 2918–32. http://dx.doi.org/10.2174/0929867325666180228165527.
Повний текст джерелаАнотація:
Some DNA or RNA sequences rich in guanine (G) nucleotides can adopt noncanonical conformations known as G-quadruplexes (G4). In the nuclear genome, G4 motifs have been associated with genome instability and gene expression defects, but they are increasingly recognized to be regulatory structures. Recent studies have revealed that G4 structures can form in the mitochondrial genome (mtDNA) and potential G4 forming sequences are associated with the origin of mtDNA deletions. However, little is known about the regulatory role of G4 structures in mitochondria. In this short review, we will explore the potential for G4 structures to regulate mitochondrial function, based on evidence from the nucleus.
9
Umar, Mubarak I., Danyang Ji, Chun-Yin Chan, and Chun Kit Kwok. "G-Quadruplex-Based Fluorescent Turn-On Ligands and Aptamers: From Development to Applications." Molecules 24, no. 13 (June 30, 2019): 2416. http://dx.doi.org/10.3390/molecules24132416.
Повний текст джерелаАнотація:
Guanine (G)-quadruplexes (G4s) are unique nucleic acid structures that are formed by stacked G-tetrads in G-rich DNA or RNA sequences. G4s have been reported to play significant roles in various cellular events in both macro- and micro-organisms. The identification and characterization of G4s can help to understand their different biological roles and potential applications in diagnosis and therapy. In addition to biophysical and biochemical methods to interrogate G4 formation, G4 fluorescent turn-on ligands can be used to target and visualize G4 formation both in vitro and in cells. Here, we review several representative classes of G4 fluorescent turn-on ligands in terms of their interaction mechanism and application perspectives. Interestingly, G4 structures are commonly identified in DNA and RNA aptamers against targets that include proteins and small molecules, which can be utilized as G4 tools for diverse applications. We therefore also summarize the recent development of G4-containing aptamers and highlight their applications in biosensing, bioimaging, and therapy. Moreover, we discuss the current challenges and future perspectives of G4 fluorescent turn-on ligands and G4-containing aptamers.
10
Xie, Xiao, Michela Zuffo, Marie-Paule Teulade-Fichou, and Anton Granzhan. "Identification of optimal fluorescent probes for G-quadruplex nucleic acids through systematic exploration of mono- and distyryl dye libraries." Beilstein Journal of Organic Chemistry 15 (August 6, 2019): 1872–89. http://dx.doi.org/10.3762/bjoc.15.183.
Повний текст джерелаАнотація:
A library of 52 distyryl and 9 mono-styryl cationic dyes was synthesized and investigated with respect to their optical properties, propensity to aggregation in aqueous medium, and capacity to serve as fluorescence “light-up” probes for G-quadruplex (G4) DNA and RNA structures. Among the 61 compounds, 57 dyes showed preferential enhancement of fluorescence intensity in the presence of one or another G4-DNA or RNA structure, while no dye displayed preferential response to double-stranded DNA or single-stranded RNA analytes employed at equivalent nucleotide concentration. Thus, preferential fluorimetric response towards G4 structures appears to be a common feature of mono- and distyryl dyes, including long-known mono-styryl dyes used as mitochondrial probes or protein stains. However, the magnitude of the G4-induced “light-up” effect varies drastically, as a function of both the molecular structure of the dyes and the nature or topology of G4 analytes. Although our results do not allow to formulate comprehensive structure–properties relationships, we identified several structural motifs, such as indole- or pyrrole-substituted distyryl dyes, as well as simple mono-stryryl dyes such as DASPMI [2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide] or its 4-isomer, as optimal fluorescent light-up probes characterized by high fluorimetric response (I/I 0 of up to 550-fold), excellent selectivity with respect to double-stranded DNA or single-stranded RNA controls, high quantum yield in the presence of G4 analytes (up to 0.32), large Stokes shift (up to 150 nm) and, in certain cases, structural selectivity with respect to one or another G4 folding topology. These dyes can be considered as promising G4-responsive sensors for in vitro or imaging applications. As a possible application, we implemented a simple two-dye fluorimetric assay allowing rapid topological classification of G4-DNA structures.
11
Deckard, Charles E., and Jonathan T. Sczepanski. "Polycomb repressive complex 2 binds RNA irrespective of stereochemistry." Chemical Communications 54, no. 85 (2018): 12061–64. http://dx.doi.org/10.1039/c8cc07433j.
Повний текст джерела
12
Li, Conghui, Honghong Wang, Zhinang Yin, Pingping Fang, Ruijing Xiao, Ying Xiang, Wen Wang, et al. "Ligand-induced native G-quadruplex stabilization impairs transcription initiation." Genome Research 31, no. 9 (August 16, 2021): 1546–60. http://dx.doi.org/10.1101/gr.275431.121.
Повний текст джерелаАнотація:
G-quadruplexes (G4s) are noncanonical DNA secondary structures formed through the self-association of guanines, and G4s are distributed widely across the genome. G4 participates in multiple biological processes including gene transcription, and G4-targeted ligands serve as potential therapeutic agents for DNA-targeted therapies. However, genome-wide studies of the exact roles of G4s in transcriptional regulation are still lacking. Here, we establish a sensitive G4-CUT&Tag method for genome-wide profiling of native G4s with high resolution and specificity. We find that native G4 signals are cell type–specific and are associated with transcriptional regulatory elements carrying active epigenetic modifications. Drug-induced promoter-proximal RNA polymerase II pausing promotes nearby G4 formation. In contrast, G4 stabilization by G4-targeted ligands globally reduces RNA polymerase II occupancy at gene promoters as well as nascent RNA synthesis. Moreover, ligand-induced G4 stabilization modulates chromatin states and impedes transcription initiation via inhibition of general transcription factors loading to promoters. Together, our study reveals a reciprocal genome-wide regulation between native G4 dynamics and gene transcription, which will deepen our understanding of G4 biology toward therapeutically targeting G4s in human diseases.
13
Falanga, Andrea P., Monica Terracciano, Giorgia Oliviero, Giovanni N. Roviello, and Nicola Borbone. "Exploring the Relationship between G-Quadruplex Nucleic Acids and Plants: From Plant G-Quadruplex Function to Phytochemical G4 Ligands with Pharmaceutic Potential." Pharmaceutics 14, no. 11 (November 4, 2022): 2377. http://dx.doi.org/10.3390/pharmaceutics14112377.
Повний текст джерелаАнотація:
G-quadruplex (G4) oligonucleotides are higher-order DNA and RNA secondary structures of enormous relevance due to their implication in several biological processes and pathological states in different organisms. Strategies aiming at modulating human G4 structures and their interrelated functions are first-line approaches in modern research aiming at finding new potential anticancer treatments or G4-based aptamers for various biomedical and biotechnological applications. Plants offer a cornucopia of phytocompounds that, in many cases, are effective in binding and modulating the thermal stability of G4s and, on the other hand, contain almost unexplored G4 motifs in their genome that could inspire new biotechnological strategies. Herein, we describe some G4 structures found in plants, summarizing the existing knowledge of their functions and biological role. Moreover, we review some of the most promising G4 ligands isolated from vegetal sources and report on the known relationships between such phytochemicals and G4-mediated biological processes that make them potential leads in the pharmaceutical sector.
14
Das, Rabindra, Edith Chevret, Vanessa Desplat, Sandra Rubio, Jean-Louis Mergny, and Jean Guillon. "Design, Synthesis and Biological Evaluation of New Substituted Diquinolinyl-Pyridine Ligands as Anticancer Agents by Targeting G-Quadruplex." Molecules 23, no. 1 (December 30, 2017): 81. http://dx.doi.org/10.3390/molecules23010081.
Повний текст джерелаАнотація:
G-quadruplexes (G4) are stacked non-canonical nucleic acid structures found in specific G-rich DNA or RNA sequences in the human genome. G4 structures are liable for various biological functions; transcription, translation, cell aging as well as diseases such as cancer. These structures are therefore considered as important targets for the development of anticancer agents. Small organic heterocyclic molecules are well known to target and stabilize G4 structures. In this article, we have designed and synthesized 2,6-di-(4-carbamoyl-2-quinolyl)pyridine derivatives and their ability to stabilize G4-structures have been determined through the FRET melting assay. It has been established that these ligands are selective for G4 over duplexes and show a preference for the parallel conformation. Next, telomerase inhibition ability has been assessed using three cell lines (K562, MyLa and MV-4-11) and telomerase activity is no longer detected at 0.1 μM concentration for the most potent ligand 1c. The most promising G4 ligands were also tested for antiproliferative activity against the two human myeloid leukaemia cell lines, HL60 and K562.
15
Marzano, Simona, Bruno Pagano, Nunzia Iaccarino, Anna Di Porzio, Stefano De Tito, Eleonora Vertecchi, Erica Salvati, Antonio Randazzo, and Jussara Amato. "Targeting of Telomeric Repeat-Containing RNA G-Quadruplexes: From Screening to Biophysical and Biological Characterization of a New Hit Compound." International Journal of Molecular Sciences 22, no. 19 (September 24, 2021): 10315. http://dx.doi.org/10.3390/ijms221910315.
Повний текст джерелаАнотація:
DNA G-quadruplex (G4) structures, either within gene promoter sequences or at telomeres, have been extensively investigated as potential small-molecule therapeutic targets. However, although G4s forming at the telomeric DNA have been extensively investigated as anticancer targets, few studies focus on the telomeric repeat-containing RNA (TERRA), transcribed from telomeres, as potential pharmacological targets. Here, a virtual screening approach to identify a library of drug-like putative TERRA G4 binders, in tandem with circular dichroism melting assay to study their TERRA G4-stabilizing properties, led to the identification of a new hit compound. The affinity of this compound for TERRA RNA and some DNA G4s was analyzed through several biophysical techniques and its biological activity investigated in terms of antiproliferative effect, DNA damage response (DDR) activation, and TERRA RNA expression in high vs. low TERRA-expressing human cancer cells. The selected hit showed good affinity for TERRA G4 and no binding to double-stranded DNA. In addition, biological assays showed that this compound is endowed with a preferential cytotoxic effect on high TERRA-expressing cells, where it induces a DDR at telomeres, probably by displacing TERRA from telomeres. Our studies demonstrate that the identification of TERRA G4-targeting drugs with potential pharmacological effects is achievable, shedding light on new perspectives aimed at discovering new anticancer agents targeting these G4 structures.
16
Prado Martins, Rodrigo, Sarah Findakly, Chrysoula Daskalogianni, Marie-Paule Teulade-Fichou, Marc Blondel, and Robin Fåhraeus. "In Cellulo Protein-mRNA Interaction Assay to Determine the Action of G-Quadruplex-Binding Molecules." Molecules 23, no. 12 (November 29, 2018): 3124. http://dx.doi.org/10.3390/molecules23123124.
Повний текст джерелаАнотація:
Protein-RNA interactions (PRIs) control pivotal steps in RNA biogenesis, regulate multiple physiological and pathological cellular networks, and are emerging as important drug targets. However, targeting of specific protein-RNA interactions for therapeutic developments is still poorly advanced. Studies and manipulation of these interactions are technically challenging and in vitro drug screening assays are often hampered due to the complexity of RNA structures. The binding of nucleolin (NCL) to a G-quadruplex (G4) structure in the messenger RNA (mRNA) of the Epstein-Barr virus (EBV)-encoded EBNA1 has emerged as an interesting therapeutic target to interfere with immune evasion of EBV-associated cancers. Using the NCL-EBNA1 mRNA interaction as a model, we describe a quantitative proximity ligation assay (PLA)-based in cellulo approach to determine the structure activity relationship of small chemical G4 ligands. Our results show how different G4 ligands have different effects on NCL binding to G4 of the EBNA1 mRNA and highlight the importance of in-cellulo screening assays for targeting RNA structure-dependent interactions.
17
Rocca, Roberta, Francesca Scionti, Matteo Nadai, Federica Moraca, Annalisa Maruca, Giosuè Costa, Raffaella Catalano, et al. "Chromene Derivatives as Selective TERRA G-Quadruplex RNA Binders with Antiproliferative Properties." Pharmaceuticals 15, no. 5 (April 28, 2022): 548. http://dx.doi.org/10.3390/ph15050548.
Повний текст джерелаАнотація:
In mammalian cells, telomerase transcribes telomeres in large G-rich non-coding RNA, known as telomeric repeat-containing RNA (TERRA), which folds into noncanonical nucleic acid secondary structures called G-quadruplexes (G4s). Since TERRA G4 has been shown to be involved in telomere length and translation regulation, it could provide valuable insight into fundamental biological processes, such as cancer growth, and TERRA G4 binders could represent an innovative strategy for cancer treatment. In this work, the three best candidates identified in our previous virtual screening campaign on bimolecular DNA/RNA G4s were investigated on the monomolecular Tel DNA and TERRA G4s by means of molecular modelling simulations and in vitro and in cell analysis. The results obtained in this work highlighted the stabilizing power of all the three candidates on TERRA G4. In particular, the two compounds characterized by a chromene scaffold were selective TERRA G4 binders, while the compound with a naphthyridine core acted as a dual Tel/TERRA G4-binder. A biophysical investigation by circular dichroism confirmed the relative stabilization efficiency of the compounds towards TERRA and Tel G4s. The TERRA G4 stabilizing hits showed good antiproliferative activity against colorectal and lung adenocarcinoma cell lines. Lead optimization to increase TERRA G4 stabilization may provide new powerful tools against cancer.
18
Wu, Guanhui, Zheng Xing, Elizabeth J. Tran, and Danzhou Yang. "DDX5 helicase resolves G-quadruplex and is involved in MYC gene transcriptional activation." Proceedings of the National Academy of Sciences 116, no. 41 (September 23, 2019): 20453–61. http://dx.doi.org/10.1073/pnas.1909047116.
Повний текст джерелаАнотація:
G-quadruplexes (G4) are noncanonical secondary structures formed in guanine-rich DNA and RNA sequences. MYC, one of the most critical oncogenes, forms a DNA G4 in its proximal promoter region (MycG4) that functions as a transcriptional silencer. However, MycG4 is highly stable in vitro and its regulatory role would require active unfolding. Here we report that DDX5, one of the founding members of the DEAD-box RNA helicase family, is extremely proficient at unfolding MycG4-DNA. Our results show that DDX5 is a highly active G4-resolvase that does not require a single-stranded overhang and that ATP hydrolysis is not directly coupled to G4-unfolding of DDX5. The chromatin binding sites of DDX5 are G-rich sequences. In cancer cells, DDX5 is enriched at the MYC promoter and activates MYC transcription. The DDX5 interaction with the MYC promoter and DDX5-mediated MYC activation is inhibited by G4-interactive small molecules. Our results uncover a function of DDX5 in resolving DNA and RNA G4s and suggest a molecular target to suppress MYC for cancer intervention.
19
Figueiredo, Joana, Tiago Santos, André Miranda, Daniela Alexandre, Bernardo Teixeira, Pedro Simões, Jéssica Lopes-Nunes, and Carla Cruz. "Ligands as Stabilizers of G-Quadruplexes in Non-Coding RNAs." Molecules 26, no. 20 (October 13, 2021): 6164. http://dx.doi.org/10.3390/molecules26206164.
Повний текст джерелаАнотація:
The non-coding RNAs (ncRNA) are RNA transcripts with different sizes, structures and biological functions that do not encode functional proteins. RNA G-quadruplexes (rG4s) have been found in small and long ncRNAs. The existence of an equilibrium between rG4 and stem−loop structures in ncRNAs and its effect on biological processes remains unexplored. For example, deviation from the stem−loop leads to deregulated mature miRNA levels, demonstrating that miRNA biogenesis can be modulated by ions or small molecules. In light of this, we report several examples of rG4s in certain types of ncRNAs, and the implications of G4 stabilization using small molecules, also known as G4 ligands, in the regulation of gene expression, miRNA biogenesis, and miRNA−mRNA interactions. Until now, different G4 ligands scaffolds were synthesized for these targets. The regulatory role of the above-mentioned rG4s in ncRNAs can be used as novel therapeutic approaches for adjusting miRNA levels.
20
Puig Lombardi, Emilia, and Arturo Londoño-Vallejo. "A guide to computational methods for G-quadruplex prediction." Nucleic Acids Research 48, no. 1 (November 22, 2019): 1–15. http://dx.doi.org/10.1093/nar/gkz1097.
Повний текст джерелаАнотація:
Abstract Guanine-rich nucleic acids can fold into the non-B DNA or RNA structures called G-quadruplexes (G4). Recent methodological developments have allowed the characterization of specific G-quadruplex structures in vitro as well as in vivo, and at a much higher throughput, in silico, which has greatly expanded our understanding of G4-associated functions. Typically, the consensus motif G3+N1–7G3+N1–7G3+N1–7G3+ has been used to identify potential G-quadruplexes from primary sequence. Since, various algorithms have been developed to predict the potential formation of quadruplexes directly from DNA or RNA sequences and the number of studies reporting genome-wide G4 exploration across species has rapidly increased. More recently, new methodologies have also appeared, proposing other estimates which consider non-canonical sequences and/or structure propensity and stability. The present review aims at providing an updated overview of the current open-source G-quadruplex prediction algorithms and straightforward examples of their implementation.
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Maltby, Connor J., James P. R. Schofield, Steven D. Houghton, Ita O’Kelly, Mariana Vargas-Caballero, Katrin Deinhardt, and Mark J. Coldwell. "A 5′ UTR GGN repeat controls localisation and translation of a potassium leak channel mRNA through G-quadruplex formation." Nucleic Acids Research 48, no. 17 (September 1, 2020): 9822–39. http://dx.doi.org/10.1093/nar/gkaa699.
Повний текст джерелаАнотація:
Abstract RNA G-quadruplexes (G4s) are secondary structures proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within some neuronal mRNAs are known to control distal localisation and local translation, contributing to distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5′ UTR of the potassium 2-pore domain leak channel Task3 mRNA. Biophysical analyses show that this (GGN)13 repeat forms a parallel G4 in vitro exhibiting the stereotypical potassium specificity of G4s, remaining thermostable under physiological ionic conditions. Through mouse brain tissue G4-RNA immunoprecipitation, we further confirm that Task3 mRNA forms a G4 structure in vivo. The G4 is inhibitory to translation of Task3 in vitro and is overcome through activity of a G4-specific helicase DHX36, increasing K+ leak currents and membrane hyperpolarisation in HEK293 cells. Further, we observe that this G4 is fundamental to ensuring delivery of Task3 mRNA to distal primary cortical neurites. It has been shown that aberrant Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is important in regulated local expression of Task3 leak channels that maintain K+ leak within neurons.
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Zaitseva, Snizhana O., Nadezhda S. Baleeva, Timofei S. Zatsepin, Ivan N. Myasnyanko, Anton V. Turaev, Galina E. Pozmogova, Alexei A. Khrulev, Anna M. Varizhuk, Mikhail S. Baranov, and Andrey V. Aralov. "Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues." Sensors 20, no. 3 (February 9, 2020): 915. http://dx.doi.org/10.3390/s20030915.
Повний текст джерелаАнотація:
Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.
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Lavigne, Marc, Olivier Helynck, Pascal Rigolet, Rofia Boudria-Souilah, Mireille Nowakowski, Bruno Baron, Sébastien Brülé, et al. "SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction." Nucleic Acids Research 49, no. 13 (July 7, 2021): 7695–712. http://dx.doi.org/10.1093/nar/gkab571.
Повний текст джерелаАнотація:
Abstract The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds.
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Dickerhoff, Jonathan, Kassandra R. Warnecke, Kaibo Wang, Nanjie Deng, and Danzhou Yang. "Evaluating Molecular Docking Software for Small Molecule Binding to G-Quadruplex DNA." International Journal of Molecular Sciences 22, no. 19 (October 6, 2021): 10801. http://dx.doi.org/10.3390/ijms221910801.
Повний текст джерелаАнотація:
G-quadruplexes are four-stranded nucleic acid secondary structures of biological significance and have emerged as an attractive drug target. The G4 formed in the MYC promoter (MycG4) is one of the most studied small-molecule targets, and a model system for parallel structures that are prevalent in promoter DNA G4s and RNA G4s. Molecular docking has become an essential tool in structure-based drug discovery for protein targets, and is also increasingly applied to G4 DNA. However, DNA, and in particular G4, binding sites differ significantly from protein targets. Here we perform the first systematic evaluation of four commonly used docking programs (AutoDock Vina, DOCK 6, Glide, and RxDock) for G4 DNA-ligand binding pose prediction using four small molecules whose complex structures with the MycG4 have been experimentally determined in solution. The results indicate that there are considerable differences in the performance of the docking programs and that DOCK 6 with GB/SA rescoring performs better than the other programs. We found that docking accuracy is mainly limited by the scoring functions. The study shows that current docking programs should be used with caution to predict G4 DNA-small molecule binding modes.
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Wang, Ju, Junqin Qiao, Weijuan Zheng, and Hongzhen Lian. "Study on the Interaction of a Peptide Targeting Specific G-Quadruplex Structures Based on Chromatographic Retention Behavior." International Journal of Molecular Sciences 24, no. 2 (January 11, 2023): 1438. http://dx.doi.org/10.3390/ijms24021438.
Повний текст джерелаАнотація:
G-quadruplexes (G4s) are of vital biological significance and G4-specific ligands with conformational selectivity show great application potential in disease treatment and biosensing. RHAU, a RNA helicase associated with AU-rich element, exerts biological functions through the mediation of G4s and has been identified to be a G4 binder. Here, we investigated the interactions between the RHAU peptide and G4s with different secondary structures using size exclusion chromatography (SEC) in association with circular dichroism (CD), ultraviolet-visible (UV-Vis) absorption, and native polyacrylamide gel electrophoresis (Native-PAGE). Spectral results demonstrated that the RHAU peptide did not break the main structure of G4s, making it more reliable for G4 structural analysis. The RHAU peptide was found to display a structural selectivity for a preferential binding to parallel G4s as reflected by the distinct chromatographic retention behaviors. In addition, the RHAU peptide exhibited different interactions with intermolecular parallel G4s and intramolecular parallel G4s, providing a novel recognition approach to G4 structures. The findings of this study enriched the insight into the binding of RHAU to G4s with various conformations. It is noteworthy that SEC technology can be easy and reliable for elucidating G4–peptide interactions, especially for a multiple G4 coexisting system, which supplied an alternative strategy to screen novel specific ligands for G4s.
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Thavornwatanayong, Thongthai, Simin Zheng, Sabine Jean Guillaume, and Bao Q. Vuong. "The DEAH-box helicase RHAU regulates immunoglobulin class switch recombination." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 112.08. http://dx.doi.org/10.4049/jimmunol.208.supp.112.08.
Повний текст джерелаАнотація:
Abstract During a humoral immune response, B cells produce alternative immunoglobulin isotypes (IgG, IgA, IgE) through class switch recombination (CSR). Initiation of CSR requires AID-mediated deamination of switch (S) regions in the immunoglobulin heavy chain (IgH) locus. Although G-quadruplex(G4)-forming S region RNAs bind AID and localize AID to S region DNA sequences, the molecular mechanism that regulates the transition of AID binding from RNA to DNA remains uncharacterized. Highly stable G4 structures necessitate helicases to unwind the G-G hydrogen bonds, which in turn may permit the base-pairing of the S transcript to the complementary DNA sequence in the IgH locus during CSR. An S region RNA pull-down assay identified the RNA Helicase associated with AU-rich element (RHAU), a DEAH-box RNA helicase, as an S transcript binding protein. To examine the role of RHAU in CSR, we genetically deleted a floxed RHAU allele in B cells of mice using CD23-Cre. Conditional deletion of RHAU in B cells reduced CSR in vivo and in vitro to approximately 50% of wild-type controls. This data suggests that RHAU plays an important role in CSR. We hypothesize that RHAU unwinds G4 in S transcripts to facilitate the handoff of AID from G4-RNA to S region DNA during CSR. Supported by The National Cancer Institute (2U54CA132378) and The National Institute of General Medical Sciences (1SC1GM132035-01)
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Sauer, Markus, and Katrin Paeschke. "G-quadruplex unwinding helicases and their function in vivo." Biochemical Society Transactions 45, no. 5 (September 22, 2017): 1173–82. http://dx.doi.org/10.1042/bst20170097.
Повний текст джерелаАнотація:
The concept that G-quadruplex (G4) structures can form within DNA or RNA in vitro has been long known and extensively discussed. In recent years, accumulating evidences imply that G-quadruplex structures form in vivo. Initially, inefficient regulation of G-quadruplex structures was mainly associated with genome instability. However, due to the location of G-quadruplex motifs and their evolutionary conservation, different cellular functions of these structures have been postulated (e.g. in telomere maintenance, DNA replication, transcription, and translation). Regardless of their function, efficient and controlled formation and unwinding are very important, because ‘mis’-regulated G-quadruplex structures are detrimental for a given process, causing genome instability and diseases. Several helicases have been shown to target and regulate specific G-quadruplex structures. This mini-review focuses on the biological consequences of G4 disruption by different helicases in vivo.
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McRae, Ewan K. S., Evan P. Booy, Gay Pauline Padilla-Meier, and Sean A. McKenna. "On Characterizing the Interactions between Proteins and Guanine Quadruplex Structures of Nucleic Acids." Journal of Nucleic Acids 2017 (2017): 1–11. http://dx.doi.org/10.1155/2017/9675348.
Повний текст джерелаАнотація:
Guanine quadruplexes (G4s) are four-stranded secondary structures of nucleic acids which are stabilized by noncanonical hydrogen bonding systems between the nitrogenous bases as well as extensive base stacking, or pi-pi, interactions. Formation of these structures in either genomic DNA or cellular RNA has the potential to affect cell biology in many facets including telomere maintenance, transcription, alternate splicing, and translation. Consequently, G4s have become therapeutic targets and several small molecule compounds have been developed which can bind such structures, yet little is known about how G4s interact with their native protein binding partners. This review focuses on the recognition of G4s by proteins and small peptides, comparing the modes of recognition that have thus far been observed. Emphasis will be placed on the information that has been gained through high-resolution crystallographic and NMR structures of G4/peptide complexes as well as biochemical investigations of binding specificity. By understanding the molecular features that lead to specificity of G4 binding by native proteins, we will be better equipped to target protein/G4 interactions for therapeutic purposes.
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Wu, Yuliang, Kazuo Shin-ya, and Robert M. Brosh. "FANCJ Helicase Defective in Fanconia Anemia and Breast Cancer Unwinds G-Quadruplex DNA To Defend Genomic Stability." Molecular and Cellular Biology 28, no. 12 (April 21, 2008): 4116–28. http://dx.doi.org/10.1128/mcb.02210-07.
Повний текст джерелаАнотація:
ABSTRACT FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.
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Kharel, Prakash, Gertraud Becker, Vladimir Tsvetkov, and Pavel Ivanov. "Properties and biological impact of RNA G-quadruplexes: from order to turmoil and back." Nucleic Acids Research 48, no. 22 (December 2, 2020): 12534–55. http://dx.doi.org/10.1093/nar/gkaa1126.
Повний текст джерелаАнотація:
Abstract Guanine-quadruplexes (G4s) are non-canonical four-stranded structures that can be formed in guanine (G) rich nucleic acid sequences. A great number of G-rich sequences capable of forming G4 structures have been described based on in vitro analysis, and evidence supporting their formation in live cells continues to accumulate. While formation of DNA G4s (dG4s) within chromatin in vivo has been supported by different chemical, imaging and genomic approaches, formation of RNA G4s (rG4s) in vivo remains a matter of discussion. Recent data support the dynamic nature of G4 formation in the transcriptome. Such dynamic fluctuation of rG4 folding-unfolding underpins the biological significance of these structures in the regulation of RNA metabolism. Moreover, rG4-mediated functions may ultimately be connected to mechanisms underlying disease pathologies and, potentially, provide novel options for therapeutics. In this framework, we will review the landscape of rG4s within the transcriptome, focus on their potential impact on biological processes, and consider an emerging connection of these functions in human health and disease.
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Bashkirov, Vladimir I., Harry Scherthan, Jachen A. Solinger, Jean-Marie Buerstedde, and Wolf-Dietrich Heyer. "A Mouse Cytoplasmic Exoribonuclease (mXRN1p) with Preference for G4 Tetraplex Substrates." Journal of Cell Biology 136, no. 4 (February 24, 1997): 761–73. http://dx.doi.org/10.1083/jcb.136.4.761.
Повний текст джерелаАнотація:
Exoribonucleases are important enzymes for the turnover of cellular RNA species. We have isolated the first mammalian cDNA from mouse demonstrated to encode a 5′–3′ exoribonuclease. The structural conservation of the predicted protein and complementation data in Saccharomyces cerevisiae suggest a role in cytoplasmic mRNA turnover and pre-rRNA processing similar to that of the major cytoplasmic exoribonuclease Xrn1p in yeast. Therefore, a key component of the mRNA decay system in S. cerevisiae has been conserved in evolution from yeasts to mammals. The purified mouse protein (mXRN1p) exhibited a novel substrate preference for G4 RNA tetraplex–containing substrates demonstrated in binding and hydrolysis experiments. mXRN1p is the first RNA turnover function that has been localized in the cytoplasm of mammalian cells. mXRN1p was distributed in small granules and was highly enriched in discrete, prominent foci. The specificity of mXRN1p suggests that RNAs containing G4 tetraplex structures may occur in vivo and may have a role in RNA turnover.
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Yoshida, Atsuhiro, Takanori Oyoshi, Akiyo Suda, Shiroh Futaki, and Miki Imanishi. "Recognition of G-quadruplex RNA by a crucial RNA methyltransferase component, METTL14." Nucleic Acids Research 50, no. 1 (December 15, 2021): 449–57. http://dx.doi.org/10.1093/nar/gkab1211.
Повний текст джерелаАнотація:
Abstract N6-methyladenosine (m6A) is an important epitranscriptomic chemical modification that is mainly catalyzed by the METTL3/METTL14 RNA methyltransferase heterodimer. Although m6A is found at the consensus sequence of 5′-DRACH-3′ in various transcripts, the mechanism by which METTL3/METTL14 determines its target is unclear. This study aimed to clarify the RNA binding property of METTL3/METTL14. We found that the methyltransferase heterodimer itself has a binding preference for RNA G-quadruplex (rG4) structures, which are non-canonical four-stranded structures formed by G-rich sequences, via the METTL14 RGG repeats. Additionally, the methyltransferase heterodimer selectively methylated adenosines close to the rG4 sequences. These results suggest a possible process for direct recruitment of METTL3/METTL14 to specific methylation sites, especially near the G4-forming regions. This study is the first to report the RNA binding preference of the m6A writer complex for the rG4 structure and provides insights into the role of rG4 in epitranscriptomic regulation.
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Roxo, Carolina, Weronika Kotkowiak, and Anna Pasternak. "G4 Matters—The Influence of G-Quadruplex Structural Elements on the Antiproliferative Properties of G-Rich Oligonucleotides." International Journal of Molecular Sciences 22, no. 9 (May 6, 2021): 4941. http://dx.doi.org/10.3390/ijms22094941.
Повний текст джерелаАнотація:
G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich sequences of DNA or RNA that have attracted increased attention as anticancer agents. This systematic study aimed to investigate the anticancer potential of five G4-forming, sequence-related DNA molecules in terms of their thermodynamic and structural properties, biostability and cellular uptake. The antiproliferative studies revealed that less thermodynamically stable G4s with three G-tetrads in the core and longer loops are more predisposed to effectively inhibit cancer cell growth. By contrast, highly structured G4s with an extended core containing four G-tetrads and longer loops are characterized by more efficient cellular uptake and improved biostability. Various analyses have indicated that the G4 structural elements are intrinsic to the biological activity of these molecules. Importantly, the structural requirements are different for efficient cancer cell line inhibition and favorable G4 cellular uptake. Thus, the ultimate antiproliferative potential of G4s is a net result of the specific balance among the structural features that are favorable for efficient uptake and those that increase the inhibitory activity of the studied molecules. Understanding the G4 structural features and their role in the biological activity of G-rich molecules might facilitate the development of novel, more potent G4-based therapeutics with unprecedented anticancer properties.
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Pirota, Valentina, Enrico Lunghi, Alessandra Benassi, Emmanuele Crespan, Mauro Freccero, and Filippo Doria. "Selective Binding and Redox-Activity on Parallel G-Quadruplexes by Pegylated Naphthalene Diimide-Copper Complexes." Molecules 26, no. 16 (August 19, 2021): 5025. http://dx.doi.org/10.3390/molecules26165025.
Повний текст джерелаАнотація:
G-quadruplexes (G4s) are higher-order supramolecular structures, biologically important in the regulation of many key processes. Among all, the recent discoveries relating to RNA-G4s, including their potential involvement as antiviral targets against COVID-19, have triggered the ever-increasing need to develop selective molecules able to interact with parallel G4s. Naphthalene diimides (NDIs) are widely exploited as G4 ligands, being able to induce and strongly stabilize these structures. Sometimes, a reversible NDI-G4 interaction is also associated with an irreversible one, due to the cleavage and/or modification of G4s by functional-NDIs. This is the case of NDI-Cu-DETA, a copper(II) complex able to cleave G4s in the closest proximity to the target binding site. Herein, we present two original Cu(II)-NDI complexes, inspired by NDI-Cu-DETA, differently functionalized with 2-(2-aminoethoxy)ethanol side-chains, to selectively drive redox-catalyzed activity towards parallel G4s. The selective interaction toward parallel G4 topology, controlled by the presence of 2-(2-aminoethoxy)ethanol side chains, was already firmly demonstrated by us using core-extended NDIs. In the present study, the presence of protonable moieties and the copper(II) cavity, increases the binding affinity and specificity of these two NDIs for a telomeric RNA-G4. Once defined the copper coordination relationship and binding constants by competition titrations, ability in G4 stabilization, and ROS-induced cleavage were analyzed. The propensity in the stabilization of parallel topology was highlighted for both of the new compounds HP2Cu and PE2Cu. The results obtained are particularly promising, paving the way for the development of new selective functional ligands for binding and destructuring parallel G4s.
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Biver, Tarita. "Discriminating between Parallel, Anti-Parallel and Hybrid G-Quadruplexes: Mechanistic Details on Their Binding to Small Molecules." Molecules 27, no. 13 (June 29, 2022): 4165. http://dx.doi.org/10.3390/molecules27134165.
Повний текст джерелаАнотація:
G-quadruplexes (G4) are now extensively recognised as a peculiar non-canonical DNA geometry that plays a prime importance role in processes of biological relevance whose number is increasing continuously. The same is true for the less-studied RNA G4 counterpart. G4s are stable structures; however, their geometrical parameters may be finely tuned not only by the presence of particular sequences of nucleotides but also by the salt content of the medium or by a small molecule that may act as a peculiar topology inducer. As far as the interest in G4s increases and our knowledge of these species deepens, researchers do not only verify the G4s binding by small molecules and the subsequent G4 stabilisation. The most innovative studies now aim to elucidate the mechanistic details of the interaction and the ability of a target species (drug) to bind only to a peculiar G4 geometry. In this focused review, we survey the advances in the studies of the binding of small molecules of medical interest to G4s, with particular attention to the ability of these species to bind differently (intercalation, lateral binding or sitting atop) to different G4 topologies (parallel, anti-parallel or hybrid structures). Some species, given the very high affinity with some peculiar G4 topology, can first bind to a less favourable geometry and then induce its conversion. This aspect is also considered.
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Bezzi, Georgina, Ernesto J. Piga, Andrés Binolfi, and Pablo Armas. "CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands." International Journal of Molecular Sciences 22, no. 5 (March 5, 2021): 2614. http://dx.doi.org/10.3390/ijms22052614.
Повний текст джерелаАнотація:
The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (−gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the −gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.
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Brázda, Václav, Yu Luo, Martin Bartas, Patrik Kaura, Otilia Porubiaková, Jiří Šťastný, Petr Pečinka, et al. "G-Quadruplexes in the Archaea Domain." Biomolecules 10, no. 9 (September 21, 2020): 1349. http://dx.doi.org/10.3390/biom10091349.
Повний текст джерелаАнотація:
The importance of unusual DNA structures in the regulation of basic cellular processes is an emerging field of research. Amongst local non-B DNA structures, G-quadruplexes (G4s) have gained in popularity during the last decade, and their presence and functional relevance at the DNA and RNA level has been demonstrated in a number of viral, bacterial, and eukaryotic genomes, including humans. Here, we performed the first systematic search of G4-forming sequences in all archaeal genomes available in the NCBI database. In this article, we investigate the presence and locations of G-quadruplex forming sequences using the G4Hunter algorithm. G-quadruplex-prone sequences were identified in all archaeal species, with highly significant differences in frequency, from 0.037 to 15.31 potential quadruplex sequences per kb. While G4 forming sequences were extremely abundant in Hadesarchaea archeon (strikingly, more than 50% of the Hadesarchaea archaeon isolate WYZ-LMO6 genome is a potential part of a G4-motif), they were very rare in the Parvarchaeota phylum. The presence of G-quadruplex forming sequences does not follow a random distribution with an over-representation in non-coding RNA, suggesting possible roles for ncRNA regulation. These data illustrate the unique and non-random localization of G-quadruplexes in Archaea.
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Miglietta, Giulia, Marco Russo, and Giovanni Capranico. "G-quadruplex–R-loop interactions and the mechanism of anticancer G-quadruplex binders." Nucleic Acids Research 48, no. 21 (November 2, 2020): 11942–57. http://dx.doi.org/10.1093/nar/gkaa944.
Повний текст джерелаАнотація:
Abstract Genomic DNA and cellular RNAs can form a variety of non-B secondary structures, including G-quadruplex (G4) and R-loops. G4s are constituted by stacked guanine tetrads held together by Hoogsteen hydrogen bonds and can form at key regulatory sites of eukaryote genomes and transcripts, including gene promoters, untranslated exon regions and telomeres. R-loops are 3-stranded structures wherein the two strands of a DNA duplex are melted and one of them is annealed to an RNA. Specific G4 binders are intensively investigated to discover new effective anticancer drugs based on a common rationale, i.e.: the selective inhibition of oncogene expression or specific impairment of telomere maintenance. However, despite the high number of known G4 binders, such a selective molecular activity has not been fully established and several published data point to a different mode of action. We will review published data that address the close structural interplay between G4s and R-loops in vitro and in vivo, and how these interactions can have functional consequences in relation to G4 binder activity. We propose that R-loops can play a previously-underestimated role in G4 binder action, in relation to DNA damage induction, telomere maintenance, genome and epigenome instability and alterations of gene expression programs.
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Masson, Thibaut, Corinne Landras Guetta, Eugénie Laigre, Anne Cucchiarini, Patricia Duchambon, Marie-Paule Teulade-Fichou, and Daniela Verga. "BrdU immuno-tagged G-quadruplex ligands: a new ligand-guided immunofluorescence approach for tracking G-quadruplexes in cells." Nucleic Acids Research 49, no. 22 (December 7, 2021): 12644–60. http://dx.doi.org/10.1093/nar/gkab1166.
Повний текст джерелаАнотація:
Abstract G-quadruplexes (G4s) are secondary structures forming in G-rich nucleic acids. G4s are assumed to play critical roles in biology, nonetheless their detection in cells is still challenging. For tracking G4s, synthetic molecules (G4 ligands) can be used as reporters and have found wide application for this purpose through chemical functionalization with a fluorescent tag. However, this approach is limited by a low-labeling degree impeding precise visualization in specific subcellular regions. Herein, we present a new visualization strategy based on the immuno-recognition of 5-bromo-2′-deoxyuridine (5-BrdU) modified G4 ligands, functionalized prior- or post-G4-target binding by CuAAC. Remarkably, recognition of the tag by antibodies leads to the detection of the modified ligands exclusively when bound to a G4 target both in vitro, as shown by ELISA, and in cells, thereby providing a highly efficient G4-ligand Guided Immunofluorescence Staining (G4-GIS) approach. The obtained signal amplification revealed well-defined fluorescent foci located in the perinuclear space and RNase treatment revealed the preferential binding to G4-RNA. Furthermore, ligand treatment affected significantly BG4 foci formation in cells. Our work headed to the development of a new imaging approach combining the advantages of immunostaining and G4-recognition by G4 ligands leading to visualization of G4/ligands species in cells with unrivaled precision and sensitivity.
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Zhu, Yance, Wenhao Liu, and Chao Zhang. "G-Quadruplexes Formation at the Upstream Region of Replication Origin (OriL) of the Pseudorabies Virus: Implications for Antiviral Targets." Viruses 13, no. 11 (November 4, 2021): 2219. http://dx.doi.org/10.3390/v13112219.
Повний текст джерелаАнотація:
Pseudorabies virus (PRV) is the causative agent of Aujeszky’s disease, which still causes large economic losses for the swine industry. Therefore, it is urgent to find a new strategy to prevent and control PRV infection. Previous studies have proven that guanine (G)-rich DNA or RNA sequences in some other viruses’ genomes have the potential to form G-quadruplex (G4), which serve as promising antivirus targets. In this study, we identified two novel G4-forming sequences, OriL-A and OriL-S, which are located at the upstream origin of replication (OriL) in the PRV genome and conserved across 32 PRV strains. Circular dichroism (CD) spectroscopy and a gel electrophoresis assay showed that the two G-rich sequences can fold into parallel G4 structures in vitro. Moreover, fluorescence resonance energy transfer (FRET) melting and a Taq polymerase stop assay indicated that the G4 ligand PhenDC3 has the capacity to bind and stabilize the G4. Notably, the treatment of PRV-infected cells with G4-stabilizer PhenDC3 significantly inhibited PRV DNA replication in host cells but did not affect PRV’s attachment and entry. These results not only expand our knowledge about the G4 characteristics in the PRV genome but also suggest that G4 may serve as an innovative therapeutic target against PRV.
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Huang, Mu-Ching, I.-Te Chu, Zi-Fu Wang, Steven Lin, Ta-Chau Chang, and Chin-Tin Chen. "A G-Quadruplex Structure in the Promoter Region of CLIC4 Functions as a Regulatory Element for Gene Expression." International Journal of Molecular Sciences 19, no. 9 (September 10, 2018): 2678. http://dx.doi.org/10.3390/ijms19092678.
Повний текст джерелаАнотація:
The differential transcriptional expression of CLIC4 between tumor cells and the surrounding stroma during cancer progression has been suggested to have a tumor-promoting effect. However, little is known about the transcriptional regulation of CLIC4. To better understand how this gene is regulated, the promoter region of CLIC4 was analyzed. We found that a high GC content near the transcriptional start site (TSS) might form an alternative G-quadruplex (G4) structure. Nuclear magnetic resonance spectroscopy (NMR) confirmed their formation in vitro. The reporter assay showed that one of the G4 structures exerted a regulatory role in gene transcription. When the G4-forming sequence was mutated to disrupt the G4 structure, the transcription activity dropped. To examine whether this G4 structure actually has an influence on gene transcription in the chromosome, we utilized the CRISPR/Cas9 system to edit the G4-forming sequence within the CLIC4 promoter in the cell genome. The pop-in/pop-out strategy was adopted to isolate the precisely-edited A375 cell clone. In CRISPR-modified A375 cell clones whose G4 was disrupted, there was a decrease in the endogenous CLIC4 messenger RNA (mRNA) expression level. In conclusion, we found that the G4 structure in the CLIC4 promoter might play an important role in regulating the level of transcription.
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Dharmaiah, Sharvari, Brandon Chen, Prit Benny Malgulwar, Vasudev Tadimeti, Ahsan Farooqi, and Jason Huse. "DNAR-11. CHARACTERIZING THE GENOMIC CONSEQUENCES OF G-QUADRUPLEX STABILIZATION IN ATRX-DEFICIENT HIGH-GRADE GLIOMA." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii92—vii93. http://dx.doi.org/10.1093/neuonc/noac209.343.
Повний текст джерелаАнотація:
Abstract α-thalassaemia/mental retardation X-linked (ATRX) mutations are a critical molecular marker for high-grade glioma (HGG). These mutations lead to accumulations of abnormal DNA secondary G-quadruplex (G4) structures, thereby inducing replication stress and DNA damage. As G4s arise at GC-rich regions (i.e., pericentromeric and telomeric regions), ATRX-deficiency alters genome-wide accessibility of chromatin and causes transcriptional dysregulation. However, the genomic consequences of this in the context of ATRX-deficiency are poorly understood. Our goal is to target ATRX deficiency through G4 stabilizers, a class of novel small molecule compounds that selectively bind to and stabilize G4s. Using a combination of functional experiments such as cell viability, western blot, flow cytometry, RNA-sequencing (RNA-seq), and immunofluorescence (IF), we evaluated the mechanisms that drive selective lethality upon G4 stabilization. Patient-derived glioma stem cells (GSCs) were treated with either vehicle (DMSO) or varying doses of CX-5461 (G4 stabilizer, Senhwa Biosciences). Excitingly, ATRX-deficient GSCs demonstrate dose-dependent enhanced sensitivity to G4 stabilization, compared to ATRX-intact and vehicle controls. Cell viability assays confirmed the specificity of CX-5461 in comparison to other commercially used G4 stabilizers. G4 stabilization activated p53-independent apoptosis and exhibited G2/M arrest in ATRX-deficient GSCs and, interestingly, upregulated expression of both ATR and ATM pathways, indicating enhanced replication stress and DNA damage, respectively. IF staining confirmed enhanced induction of replication stress and DNA damage markers 53BP1 and gH2AX. Our preliminary findings suggest that ATR and ATM activation leads to Cyclin D1 degradation and inhibition of transcription factor NF-κB, thereby driving apoptosis. In fact, RNA-seq analyses revealed positive enrichment of apoptosis, DNA repair, and NF-κB pathways and negative enrichment of the G2/M checkpoint in ATRX-deficient GSCs treated with CX-5461. Our work defines mechanisms of action and efficacy of a novel therapeutic strategy for pre-clinical ATRX-deficient HGG models, with strong implications for other ATRX-deficient cancers and potential translation into clinical practice.
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Cadoni, Enrico, Lessandro De Paepe, Alex Manicardi, and Annemieke Madder. "Beyond small molecules: targeting G-quadruplex structures with oligonucleotides and their analogues." Nucleic Acids Research 49, no. 12 (May 12, 2021): 6638–59. http://dx.doi.org/10.1093/nar/gkab334.
Повний текст джерелаАнотація:
Abstract G-Quadruplexes (G4s) are widely studied secondary DNA/RNA structures, naturally occurring when G-rich sequences are present. The strategic localization of G4s in genome areas of crucial importance, such as proto-oncogenes and telomeres, entails fundamental implications in terms of gene expression regulation and other important biological processes. Although thousands of small molecules capable to induce G4 stabilization have been reported over the past 20 years, approaches based on the hybridization of a synthetic probe, allowing sequence-specific G4-recognition and targeting are still rather limited. In this review, after introducing important general notions about G4s, we aim to list, explain and critically analyse in more detail the principal approaches available to target G4s by using oligonucleotides and synthetic analogues such as Locked Nucleic Acids (LNAs) and Peptide Nucleic Acids (PNAs), reporting on the most relevant examples described in literature to date.
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Aubets, Eva, Alex J. Félix, Miguel Garavís, Laura Reyes, Anna Aviñó, Ramón Eritja, Carlos J. Ciudad, and Véronique Noé. "Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins." International Journal of Molecular Sciences 21, no. 14 (July 16, 2020): 5028. http://dx.doi.org/10.3390/ijms21145028.
Повний текст джерелаАнотація:
Thymidylate synthase (TYMS) enzyme is an anti-cancer target given its role in DNA biosynthesis. TYMS inhibitors (e.g., 5-Fluorouracil) can lead to drug resistance through an autoregulatory mechanism of TYMS that causes its overexpression. Since G-quadruplexes (G4) can modulate gene expression, we searched for putative G4 forming sequences (G4FS) in the TYMS gene that could be targeted using polypurine reverse Hoogsteen hairpins (PPRH). G4 structures in the TYMS gene were detected using the quadruplex forming G-rich sequences mapper and confirmed through spectroscopic approaches such as circular dichroism and NMR using synthetic oligonucleotides. Interactions between G4FS and TYMS protein or G4FS and a PPRH targeting this sequence (HpTYMS-G4-T) were studied by EMSA and thioflavin T staining. We identified a G4FS in the 5’UTR of the TYMS gene in both DNA and RNA capable of interacting with TYMS protein. The PPRH binds to its corresponding target dsDNA, promoting G4 formation. In cancer cells, HpTYMG-G4-T decreased TYMS mRNA and protein levels, leading to cell death, and showed a synergic effect when combined with 5-fluorouracil. These results reveal the presence of a G4 motif in the TYMS gene, probably involved in the autoregulation of TYMS expression, and the therapeutic potential of a PPRH targeted to the G4FS.
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Yamaguchi, Katsutoshi, Kiryu Asakura, Masataka Imamura, Gota Kawai, Taiichi Sakamoto, Tomomi Furihata, Robert J. Linhardt, Kazuei Igarashi, Toshihiko Toida, and Kyohei Higashi. "Polyamines stimulate the CHSY1 synthesis through the unfolding of the RNA G-quadruplex at the 5′-untraslated region." Biochemical Journal 475, no. 23 (December 6, 2018): 3797–812. http://dx.doi.org/10.1042/bcj20180672.
Повний текст джерелаАнотація:
Glycosaminoglycans (GAGs), a group of structurally related acidic polysaccharides, are primarily found as glycan moieties of proteoglycans (PGs). Among these, chondroitin sulfate (CS) and dermatan sulfate, side chains of PGs, are widely distributed in animal kingdom and show structural variations, such as sulfation patterns and degree of epimerization, which are responsible for their physiological functions through interactions with growth factors, chemokines and adhesion molecules. However, structural changes in CS, particularly the ratio of 4-O-sulfation to 6-O-sulfation (4S/6S) and CS chain length that occur during the aging process, are not fully understood. We found that 4S/6S ratio and molecular weight of CS were decreased in polyamine-depleted cells. In addition, decreased levels of chondroitin synthase 1 (CHSY1) and chondroitin 4-O-sulfotransferase 2 proteins were also observed on polyamine depletion. Interestingly, the translation initiation of CHSY1 was suppressed by a highly structured sequence (positions −202 to −117 relative to the initiation codon) containing RNA G-quadruplex (G4) structures in 5′-untranslated region. The formation of the G4s was influenced by the neighboring sequences to the G4s and polyamine stimulation of CHSY1 synthesis disappeared when the formation of the G4s was inhibited by site-directed mutagenesis. These results suggest that the destabilization of G4 structures by polyamines stimulates CHSY1 synthesis and, at least in part, contribute to the maturation of CS chains.
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Galati, Elena, Maria C. Bosio, Daniele Novarina, Matteo Chiara, Giulia M. Bernini, Alessandro M. Mozzarelli, Maria L. García-Rubio, et al. "VID22 counteracts G-quadruplex-induced genome instability." Nucleic Acids Research 49, no. 22 (December 6, 2021): 12785–804. http://dx.doi.org/10.1093/nar/gkab1156.
Повний текст джерелаАнотація:
Abstract Genome instability is a condition characterized by the accumulation of genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways affecting endogenous DNA damage and genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, reported to be involved in DNA double-strand break repair. vid22Δ cells exhibit increased levels of endogenous DNA damage, chronic DNA damage response activation and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. Vid22 binds to and protects DNA at G4-containing regions both in vitro and in vivo. Loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. Moreover, the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.
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Dickerhoff, Jonathan, Jixun Dai, and Danzhou Yang. "Structural recognition of the MYC promoter G-quadruplex by a quinoline derivative: insights into molecular targeting of parallel G-quadruplexes." Nucleic Acids Research 49, no. 10 (May 12, 2021): 5905–15. http://dx.doi.org/10.1093/nar/gkab330.
Повний текст джерелаАнотація:
Abstract DNA G-Quadruplexes (G4s) formed in oncogene promoters regulate transcription. The oncogene MYC promoter G4 (MycG4) is the most prevalent G4 in human cancers. However, the most studied MycG4 sequence bears a mutated 3′-residue crucial for ligand recognition. Here, we report a new drug-like small molecule PEQ without a large aromatic moiety that specifically binds MycG4. We determined the NMR solution structures of the wild-type MycG4 and its 2:1 PEQ complex, as well as the structure of the 2:1 PEQ complex of the widely used mutant MycG4. Comparison of the two complex structures demonstrates specific molecular recognition of MycG4 and shows the clear effect of the critical 3′-mutation on the drug binding interface. We performed a systematic analysis of the four available complex structures involving the same mutant MycG4, which can be considered a model system for parallel G4s, and revealed for the first time that the flexible flanking residues are recruited in a conserved and sequence-specific way, as well as unused potential for selective ligand-G4 hydrogen-bond interactions. Our results provide the true molecular basis for MycG4-targeting drugs and new critical insights into future rational design of drugs targeting MycG4 and parallel G4s that are prevalent in promoter and RNA G4s.
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Roach, Ruby J., Miguel Garavís, Carlos González, Geoffrey B. Jameson, Vyacheslav V. Filichev та Tracy K. Hale. "Heterochromatin protein 1α interacts with parallel RNA and DNA G-quadruplexes". Nucleic Acids Research 48, № 2 (4 грудня 2019): 682–93. http://dx.doi.org/10.1093/nar/gkz1138.
Повний текст джерелаАнотація:
Abstract The eukaryotic genome is functionally organized into domains of transcriptionally active euchromatin and domains of highly compact transcriptionally silent heterochromatin. Heterochromatin is constitutively assembled at repetitive elements that include the telomeres and centromeres. The histone code model proposes that HP1α forms and maintains these domains of heterochromatin through the interaction of its chromodomain with trimethylated lysine 9 of histone 3, although this interaction is not the sole determinant. We show here that the unstructured hinge domain, necessary for the targeting of HP1α to constitutive heterochromatin, recognizes parallel G-quadruplex (G4) assemblies formed by the TElomeric Repeat-containing RNA (TERRA) transcribed from the telomere. This provides a mechanism by which TERRA can lead to the enrichment of HP1α at telomeres to maintain heterochromatin. Furthermore, we show that HP1α binds with a faster association rate to DNA G4s of parallel topology compared to antiparallel G4s that bind slowly or not at all. Such G4–DNAs are found in the regulatory regions of several oncogenes. This implicates specific non-canonical nucleic acid structures as determinants of HP1α function and thus RNA and DNA G4s need to be considered as contributors to chromatin domain organization and the epigenome.
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Marquevielle, Julien, Coralie Robert, Olivier Lagrabette, Mona Wahid, Anne Bourdoncle, Luigi E. Xodo, Jean-Louis Mergny, and Gilmar F. Salgado. "Structure of two G-quadruplexes in equilibrium in the KRAS promoter." Nucleic Acids Research 48, no. 16 (May 20, 2020): 9336–45. http://dx.doi.org/10.1093/nar/gkaa387.
Повний текст джерелаАнотація:
Abstract KRAS is one of the most mutated oncogenes and still considered an undruggable target. An alternative strategy would consist in targeting its gene rather than the protein, specifically the formation of G-quadruplexes (G4) in its promoter. G4 are secondary structures implicated in biological processes, which can be formed among G-rich DNA (or RNA) sequences. Here we have studied the major conformations of the commonly known KRAS 32R, or simply 32R, a 32 residue sequence within the KRAS Nuclease Hypersensitive Element (NHE) region. We have determined the structure of the two major stable conformers that 32R can adopt and which display slow equilibrium (>ms) with each other. By using different biophysical methods, we found that the nucleotides G9, G25, G28 and G32 are particularly implicated in the exchange between these two conformations. We also showed that a triad at the 3′ end further stabilizes one of the G4 conformations, while the second conformer remains more flexible and less stable.
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Xu, Ying-Zhi, Thomas Raney, and Samantha L. Kendrick. "Discovery of DNA G-Quadruplexes As a New Target for B-Cell Receptor Signaling Inhibition in Diffuse Large B-Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 3502. http://dx.doi.org/10.1182/blood-2018-99-116793.
Повний текст джерелаАнотація:
Abstract Extensive gene expression profiling and RNA interference studies revealed the frequently chemo-resistant activated B-cell-like (ABC) subtype of diffuse large B-cell lymphoma (DLBCL) relies on constitutive B-cell receptor (BCR) signaling. As such, the clinical importance of BCR signaling inhibition is well appreciated and thus far led to the development of kinase and protease inhibitors. However, this therapeutic approach fails to achieve complete, sustained responses in DLBCL patients because of inherent resistance due to additional genetic lesions in other components of the BCR pathway or acquired kinase mutations. The emerging field of DNA secondary structures support that guanine (G)-rich stretches of DNA capable of adopting G-quadruplex (G4) motifs act as transcription regulatory units, or switches, that can turn gene expression on or off. Targeting G4s is likely to overcome activating kinase mutations by limiting the amount of gene available for translation into protein. Here, we explore a drug discovery effort based on targeting G4 within BCR genes critical for ABC DLBCL cell survival, CD79A, CD79B, CARD11, and MYD88. We first interrogated the BCR-related genes within the hg19 human reference genome for G-rich DNA using a G4 algorithm and discovered each of the four genes contain G4 forming sequences near promoter regions. These G4 elements formed stable G4 structures as determined by circular dichroism (CD) spectroscopy, the standard for visualizing macromolecule secondary structure formation. Melting curves are also generated from CD spectroscopy to determine the thermal stability of a given structure. The CD79A, CD79B, CARD11, and MYD88 G-rich sequences displayed classic, stable G4 structure spectra consisting of negative minima absorption peaks at 240-265 nm and a positive maximum at 260-295 nm with melting temperatures ranging from 62 to 95 °C. We then developed a high-throughput screening assay based on fluorescence resonance energy transfer (FRET) to identify G4 interactive compounds from the NCI Diversity Set IV library (1584 compounds) that uniquely interact with each of the BCR G4 sequences. This screen used the BCR G4 sequences as molecular bait where the 5´-end and 3´-end of the oligomers were labeled with a FAM- and a TAMRA-fluorophore, respectively, such that G4 formation leads to an increase in fluorescence emission (Figure 1). The initial FRET screen tested compounds at a 1:5 molecular ratio of probe to compound and measured the change in fluorescence relative to probe alone. Overall, the screen resulted in a ~1% "hit" rate for each BCR target, except for CD79B, which yielded a lower percent of interactive compounds (0.3%). Seven compounds, which included ellipticine, quinoline, and daunomycin derivatives, were identified to selectively target the CARD11 (n=3), MYD88 (n=3), or CD79A (n=1) G4s relative to other G4, single-stranded, and double-stranded DNA. Of note, all five compounds found to interact with the CD79B G4 also altered FRET of the other BCR G4 sequences. Subsequent FRET validation and CD analyses where each of the BCR sequences was incubated with increasing concentrations of candidate compounds demonstrated dose-dependent effects on G4 structure formation, particularly stabilization of the CARD11 G4 with compound NCI 9037 that resulted in a 300% FRET increase and an 8 °C shift in melting temperature at a 1:10 ratio. This study identifies DNA G4 as a new class of molecular targets for inhibiting an important oncogenic pathway. Discovery of selective compounds in addition to those with "pan" interaction, suggests the CARD11, MYD88, and CD79A G4 have unique folding patterns whereas the CD79B G4 may exhibit more common structural features. These compounds will be used as molecular tools to provide further insight into the structures and mechanisms in which G4 regulate gene transcription. In establishing a high-throughput screen, we discovered compounds for which preclinical development is ongoing and includes evaluation of the effects on BCR target gene and protein expression, inhibition of downstream BCR signaling, and consequent ABC DLBCL tumor growth and survival. This treatment strategy has high potential for leading to a breakthrough in effectively targeting the constitutively active molecules and greatly impacting the clinical management of patients with BCR-dependent DLBCL. Disclosures No relevant conflicts of interest to declare.