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The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (30 September 2020) | Viewed by 31289

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Prof. Dr. Aldo Galeone

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Department of Pharmacy, Università degli Studi di Napoli Federico II, 80131 Naples, Italy
Interests: G-quadruplex; nucleic acid secondary structures; aptamers; nucleic acid chemistry
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Dr. Veronica Esposito

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Department of Pharmacy, Università degli Studi di Napoli Federico II, 80131 Naples, Italy
Interests: G-quadruplex; aptamers; nucleic acid chemistry; nuclear magnetic resonance; molecular modelling; circular dichroism
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Dr. Antonella Virgilio

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Department of Pharmacy, Università degli Studi di Napoli Federico II, 80131 Naples, Italy
Interests: G-quadruplex; aptamers; nucleic acid chemistry; circular dichroism; electrophoresis
Special Issues, Collections and Topics in MDPI journals

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Dear Colleagues,

G-quadruplex structures are secondary conformations of nucleic acids, whose constitutive unit is the G-tetrad or G-quartet. This building block consists of a square planar arrangement of four guanosines, in which each base is associated to the adjacent ones through four hydrogen bonds. The stacking of two or more G-tetrad units can form larger and more stable structures. The occurrance of monovalent cations, between two adjacent G-tetrads or also in the center of a G-tetrad, further contributes to the structural stability of the G-quadruplex complexes. The biological significance of these DNA or RNA structures is witnessed by their occurrence or potential formation in several regions of the human genome, such as telomeres, genes promoters, and transcription start sites. Furthermore, they can be involved in the regulation of gene expression and telomere maintenance. However, the importance of the G-quadruplex structures is not confined to genetics and molecular biological research. In fact, thanks to their remarkable stability and outstanding variability, these structures constitute the scaffolds of several DNA or RNA aptamers, with important applications in pharmaceutics, analytics, and diagnostics. Furthermore, suitable G-quadruplexes are also endowed with catalytic properties. Moreover, considering their self-assembly properties, G-quadruplexes are often exploited in building nanostructures and in developing nanodevices.

This Special Issue will concern a selection of original research, review articles, and commentaries focused on diverse topics, with particular attention on the relationship between the structural features of the G-quadruplexes and their functional role.

Dr. Aldo Galeone
Prof. Veronica Esposito
Prof. Antonella Virgilio
Guest Editors

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Keywords

G-quadruplex thermodynamics
G-quadruplex CD and fluorescence spectroscopy
G-quadruplex synthesis
G-quadruplex properties
G-quadruplex structure
G-quadruplex dynamics
G-quadruplex folding
G-quadruplex biological functions
G-quadruplex aptamers
G-quadruplex as a therapeutic target
Catalytic G-quadruplexes

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The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity-2nd Edition in International Journal of Molecular Sciences (13 articles)

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20 pages, 1981 KiB

Open AccessArticle

PhenQE8, a Novel Ligand of the Human Telomeric Quadruplex

by Patricia B. Gratal, Julia G. Quero, Adrián Pérez-Redondo, Zoila Gándara and Lourdes Gude

Int. J. Mol. Sci. 2021, 22(2), 749; https://doi.org/10.3390/ijms22020749 - 13 Jan 2021

Cited by 4 | Viewed by 2863

Abstract

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

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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13 pages, 1860 KiB

Open AccessArticle

KRAS Promoter G-Quadruplexes from Sequences of Different Length: A Physicochemical Study

by Federica D’Aria, Bruno Pagano, Luigi Petraccone and Concetta Giancola

Int. J. Mol. Sci. 2021, 22(1), 448; https://doi.org/10.3390/ijms22010448 - 05 Jan 2021

Cited by 7 | Viewed by 3086

Abstract

DNA G-quadruplexes (G4s) form in relevant genomic regions and intervene in several biological processes, including the modulation of oncogenes expression, and are potential anticancer drug targets. The human KRAS proto-oncogene promoter region contains guanine-rich sequences able to fold into G4 structures. Here, by using circular dichroism and differential scanning calorimetry as complementary physicochemical methodologies, we compared the thermodynamic stability of the G4s formed by a shorter and a longer version of the KRAS promoter sequence, namely 5′-AGGGCGGTGTGGGAATAGGGAA-3′ (KRAS 22RT) and 5′-AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG-3′ (KRAS 32R). Our results show that the unfolding mechanism of KRAS 32R is more complex than that of KRAS 22RT. The different thermodynamic stability is discussed based on the recently determined NMR structures. The binding properties of TMPyP4 and BRACO-19, two well-known G4-targeting anticancer compounds, to the KRAS G4s were also investigated. The present physicochemical study aims to help in choosing the best G4 target for potential anticancer drugs. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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23 pages, 2794 KiB

Open AccessArticle

Tuning G-Quadruplex Nanostructures with Lipids. Towards Designing Hybrid Scaffolds for Oligonucleotide Delivery

by Santiago Grijalvo, Anna Clua, Marc Eres, Raimundo Gargallo and Ramon Eritja

Int. J. Mol. Sci. 2021, 22(1), 121; https://doi.org/10.3390/ijms22010121 - 24 Dec 2020

Cited by 3 | Viewed by 2318

Abstract

Two G-quadruplex forming oligonucleotides [d(TG₄T)₄ and d(TG₆T)₄] were selected as two tetramolecular quadruplex nanostructures because of their demonstrated ability to be modified with hydrophobic molecules. This allowed us to synthesize two series of G-quadruplex conjugates that differed in the number of G-tetrads, as well as in the terminal position of the lipid modification. Both solution and solid-phase syntheses were carried out to yield the corresponding lipid oligonucleotide conjugates modified at their 3′- and 5′-termini, respectively. Biophysical studies confirmed that the presence of saturated alkyl chains with different lengths did not affect the G-quadruplex integrity, but increased the stability. Next, the G-quadruplex domain was added to an 18-mer antisense oligonucleotide. Gene silencing studies confirmed the ability of such G-rich oligonucleotides to facilitate the inhibition of target Renilla luciferase without showing signs of toxicity in tumor cell lines. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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20 pages, 3548 KiB

Open AccessArticle

Spectroscopic Properties of Two 5′-(4-Dimethylamino)Azobenzene Conjugated G-Quadruplex Forming Oligonucleotides

by Concetta Imperatore, Antonio Varriale, Elisa Rivieccio, Angela Pennacchio, Maria Staiano, Sabato D’Auria, Marcello Casertano, Carlo Altucci, Mohammadhassan Valadan, Manjot Singh, Marialuisa Menna and Michela Varra

Int. J. Mol. Sci. 2020, 21(19), 7103; https://doi.org/10.3390/ijms21197103 - 26 Sep 2020

Cited by 5 | Viewed by 2588

Abstract

The synthesis of two 5′-end (4-dimethylamino)azobenzene conjugated G-quadruplex forming aptamers, the thrombin binding aptamer (TBA) and the HIV-1 integrase aptamer (T30695), was performed. Their structural behavior was investigated by means of UV, CD, fluorescence spectroscopy, and gel electrophoresis techniques in K⁺-containing buffers and water-ethanol blends. Particularly, we observed that the presence of the 5′-(4-dimethylamino)azobenzene moiety leads TBA to form multimers instead of the typical monomolecular chair-like G-quadruplex and almost hampers T30695 G-quadruplex monomers to dimerize. Fluorescence studies evidenced that both the conjugated G-quadruplexes possess unique fluorescence features when excited at wavelengths corresponding to the UV absorption of the conjugated moiety. Furthermore, a preliminary investigation of the trans-cis conversion of the dye incorporated at the 5′-end of TBA and T30695 showed that, unlike the free dye, in K⁺-containing water-ethanol-triethylamine blend the trans-to-cis conversion was almost undetectable by means of a standard UV spectrophotometer. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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21 pages, 12533 KiB

Open AccessArticle

Role of Poly [ADP-ribose] Polymerase 1 in Activating the Kirsten ras (KRAS) Gene in Response to Oxidative Stress

by Giorgio Cinque, Annalisa Ferino, Erik B. Pedersen and Luigi E. Xodo

Int. J. Mol. Sci. 2020, 21(17), 6237; https://doi.org/10.3390/ijms21176237 - 28 Aug 2020

Cited by 11 | Viewed by 2810

Abstract

In pancreatic Panc-1 cancer cells, an increase of oxidative stress enhances the level of 7,8-dihydro-8-oxoguanine (8OG) more in the KRAS promoter region containing G4 motifs than in non-G4 motif G-rich genomic regions. We found that H₂O₂ stimulates the recruitment to the KRAS promoter of poly [ADP-ribose] polymerase 1 (PARP-1), which efficiently binds to local G4 structures. Upon binding to G4 DNA, PARP-1 undergoes auto PARylation and thus becomes negatively charged. In our view this should favor the recruitment to the KRAS promoter of MAZ and hnRNP A1, as these two nuclear factors, because of their isoelectric points >7, are cationic in nature under physiological conditions. This is indeed supported by pulldown assays which showed that PARP-1, MAZ, and hnRNP A1 form a multiprotein complex with an oligonucleotide mimicking the KRAS G4 structure. Our data suggest that an increase of oxidative stress in Panc-1 cells activates a ROS-G4-PARP-1 axis that stimulates the transcription of KRAS. This mechanism is confirmed by the finding that when PARP-1 is silenced by siRNA or auto PARylation is inhibited by Veliparib, the expression of KRAS is downregulated. When Panc-1 cells are treated with H₂O₂ instead, a strong up-regulation of KRAS transcription is observed. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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15 pages, 1928 KiB

Open AccessArticle

Diversity of Parallel Guanine Quadruplexes Induced by Guanine Substitutions

by Klára Bednářová, Michaela Vorlíčková and Daniel Renčiuk

Int. J. Mol. Sci. 2020, 21(17), 6123; https://doi.org/10.3390/ijms21176123 - 25 Aug 2020

Cited by 1 | Viewed by 1765

Abstract

Recently, we reported an inhibitory effect of guanine substitutions on the conformational switch from antiparallel to parallel quadruplexes (G4) induced by dehydrating agents. As a possible cause, we proposed a difference in the sensitivity of parallel and antiparallel quadruplexes to the guanine substitutions in the resulting thermodynamic stability. Reports on the influence of guanine substitutions on the biophysical properties of intramolecular parallel quadruplexes are rare. Moreover, such reports are often complicated by the multimerisation tendencies of parallel quadruplexes. To address this incomplete knowledge, we employed circular dichroism spectroscopy (CD), both as stopped-flow-assisted fast kinetics measurements and end-point measurements, accompanied by thermodynamic analyses, based on UV absorption melting profiles, and electrophoretic methods. We showed that parallel quadruplexes are significantly more sensitive towards guanine substitutions than antiparallel ones. Furthermore, guanine-substituted variants, which in principle might correspond to native genomic sequences, distinctly differ in their biophysical properties, indicating that the four guanines in each tetrad of parallel quadruplexes are not equal. In addition, we were able to distinguish by CD an intramolecular G4 from intermolecular ones resulting from multimerisation mediated by terminal tetrad association, but not from intermolecular G4s formed due to inter-strand Hoogsteen hydrogen bond formation. In conclusion, our study indicates significant variability in parallel quadruplex structures, otherwise disregarded without detailed experimental analysis. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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13 pages, 1928 KiB

Open AccessArticle

Probing the Importance of the G-Quadruplex Grooves for the Activity of the Anti-HIV-Integrase Aptamer T30923

by Veronica Esposito, Francesca Esposito, Antonietta Pepe, Isabel Gomez Monterrey, Enzo Tramontano, Luciano Mayol, Antonella Virgilio and Aldo Galeone

Int. J. Mol. Sci. 2020, 21(16), 5637; https://doi.org/10.3390/ijms21165637 - 06 Aug 2020

Cited by 3 | Viewed by 2020

Abstract

In this paper, we report studies concerning four variants of the G-quadruplex forming anti-HIV-integrase aptamer T30923, in which specific 2′-deoxyguanosines have been singly replaced by 8-methyl-2′-deoxyguanosine residues, with the aim to exploit the methyl group positioned in the G-quadruplex grooves as a steric probe to investigate the interaction aptamer/target. Although, the various modified aptamers differ in the localization of the methyl group, NMR, circular dichroism (CD), electrophoretic and molecular modeling data suggest that all of them preserve the ability to fold in a stable dimeric parallel G-quadruplex complex resembling that of their natural counterpart T30923. However, the biological data have shown that the T30923 variants are characterized by different efficiencies in inhibiting the HIV-integrase, thus suggesting the involvement of the G-quadruplex grooves in the aptamer/target interaction. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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22 pages, 3326 KiB

Open AccessArticle

Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

by Eva Aubets, Alex J. Félix, Miguel Garavís, Laura Reyes, Anna Aviñó, Ramón Eritja, Carlos J. Ciudad and Véronique Noé

Int. J. Mol. Sci. 2020, 21(14), 5028; https://doi.org/10.3390/ijms21145028 - 16 Jul 2020

Cited by 7 | Viewed by 2958

Abstract

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. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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24 pages, 5040 KiB

Open AccessArticle

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs

by Claudia Riccardi, Domenica Musumeci, Chiara Platella, Rosa Gaglione, Angela Arciello and Daniela Montesarchio

Int. J. Mol. Sci. 2020, 21(6), 1963; https://doi.org/10.3390/ijms21061963 - 13 Mar 2020

Cited by 13 | Viewed by 3175

Abstract

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF₁₆₅ binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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Epigenetic Modulation of Chromatin States and Gene Expression by G-Quadruplex Structures

by Chiara Reina and Vincenzo Cavalieri

Int. J. Mol. Sci. 2020, 21(11), 4172; https://doi.org/10.3390/ijms21114172 - 11 Jun 2020

Cited by 23 | Viewed by 6507

Abstract

G-quadruplexes are four-stranded helical nucleic acid structures formed by guanine-rich sequences. A considerable number of studies have revealed that these noncanonical structural motifs are widespread throughout the genome and transcriptome of numerous organisms, including humans. In particular, G-quadruplexes occupy strategic locations in genomic DNA and both coding and noncoding RNA molecules, being involved in many essential cellular and organismal functions. In this review, we first outline the fundamental structural features of G-quadruplexes and then focus on the concept that these DNA and RNA structures convey a distinctive layer of epigenetic information that is critical for the complex regulation, either positive or negative, of biological activities in different contexts. In this framework, we summarize and discuss the proposed mechanisms underlying the functions of G-quadruplexes and their interacting factors. Furthermore, we give special emphasis to the interplay between G-quadruplex formation/disruption and other epigenetic marks, including biochemical modifications of DNA bases and histones, nucleosome positioning, and three-dimensional organization of chromatin. Finally, epigenetic roles of RNA G-quadruplexes in post-transcriptional regulation of gene expression are also discussed. Undoubtedly, the issues addressed in this review take on particular importance in the field of comparative epigenetics, as well as in translational research. Full article

(This article belongs to the Special Issue The Polymorphic World of G-Quadruplexes: From Structural Insights to Functional Activity)

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