G-quadruplex Formation Research Articles

G-Quadruplexes at Telomeres: Friend or Foe?

Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures.

G-quadruplex 2′-F-modified RNA aptamers targeting hemoglobin: Structure studies and colorimetric assays

Biosensors that rely on aptamers as analyte-recognizing elements (also known as aptasensors) are gaining in popularity during recent years for analytical and biomedical applications. Among them, colorimetric ELISA-like systems seem very promising for biomarker detection in medical diagnostics. For their development, one should thoroughly consider the characteristics of the aptamers, with a particular focus on the secondary structure. In this study, we performed an in-depth structural study of previously selected hemoglobin-binding 2′-F-RNA aptamers using CD spectroscopy, enzymatic probing, and specific fluorophore binding. Only a combination of different assays allowed us to prove G-quadruplex formation for anti-hemoglobin 2′-F-RNA aptamers. We also demonstrated a possible application of these 2′-F-RNA aptamers for microplate colorimetric detection of human hemoglobin in both direct and sandwich formats.

Anti-cancer activity of benzoxazinone derivatives via targeting c-Myc G-quadruplex structure

AimsThis study aimed to analyze the impact of four synthesized benzoxazinone derivatives as screening drugs on c-Myc-overexpressed cancer cells (H7402, HeLa, SK-RC-42, SGC7901, and A549) and to explore their interaction mechanisms in detail. Materials and methodsUsing morphological analysis, real-time cytotoxicity analysis, wound healing assay, reverse transcription PCR, electrophoretic mobility shift assay, and circular dichroism spectroscopy techniques. Key findingsResults revealed that these four compounds could inhibit proliferation of SK-RC-42, SGC7901, and A549 cells in five cancer cell lines to varying degrees and significantly hinder migration. More importantly, the RT-PCR assay showed that the compounds could surprisingly downregulate the expression of c-Myc mRNA in a dose-dependent manner in the five cancer cells, which may be one of the causes of cancer cell proliferation in vitro inhibition. Further EMSA assays demonstrated that at the molecular level of DNA, four compounds can induce the formation of G-quadruplexes (G4-DNAs) in the c-Myc gene promoter. In addition, the CD result of compound 1 clearly indicates that it specifically induces a c-Myc GC-rich 36mer double-stranded DNA in the c-Myc promoter to form a G-quadruplex hybrid configuration. In conclusion, the compounds studied could dose-dependently inhibit the growth and migration of the cancer cells being investigated. This is positively associated with the reduction of overexpression of the c-Myc gene, which may be significantly regulated by the association of compounds with the G-quadruplexes produced in the c-Myc gene promoter region. SignificanceWe conclude that three compounds merit further study, particularly against non-small-cell lung cancer, as leading compounds of anticancer drugs.

Versatile Cell-Specific Ligand Arrangement System onto Desired Compartments of Biodegradable Matrices for Site-Selective Cell Adhesion Using DNA Tags.

A promising approach for the regeneration of tissues or organs with three-dimensional hierarchical structures is the preparation of scaffold-cell complexes that mimic these hierarchical structures. This requires an effective technique for immobilizing cell-specific ligands at arbitrarily chosen positions on matrices. Here, we report a versatile system for arranging cell-specific ligands onto desired compartments of biodegradable matrices for site-selective cell arrangement. We utilized the specific binding abilities of specific DNAs, immobilizing them as tags to arrange cell-recognition ligands at desired areas of the matrices by specific binding with cell-recognition ligand-DNA conjugates. We synthesized poly(l-lactide) (PLLA), a biodegradable polymer, with an oligo-DNA (trimer of deoxyguanosine: dG3) attached via a poly(ethylene glycol) (PEG) spacer to generate dG3-PEG-b-PLLA. The peptides Arg-Gly-Asp-Ser (RGDS) and Arg-Glu-Asp-Val (REDV) were chosen as cell-recognition ligands and were attached to an adapter DNA (aDNA), which can specifically bind to the dG3 moiety through G-quadruplex formation. The obtained dG3-PEG-b-PLLA was deposited on a small spot of the PLLA film, and the aDNA-RGDS or aDNA-REDV conjugate was added on the film to immobilize these ligands at the spot. We confirmed the specific adhesion of L929 cells (a mouse fibroblast cell line) and human umbilical vein endothelial cells (HUVECs) on the small areas coated with dG3-PEG-b-PLLA in the presence of aDNA-RGDS and aDNA-REDV, respectively, even after applying shear stress by flowing medium across the spot. Cell-specific attachment of the target cells was effectively achieved in a spatially controlled manner. This technique has the potential for the construction of cell-scaffold complexes that mimic the hierarchical structures of natural organs and may represent a breakthrough in realizing regenerative medicine and tissue engineering of complex organs.

Single-molecule visualization of DNA G-quadruplex formation in live cells.

Substantial evidence now exists to support that formation of DNA G-quadruplexes (G4s) can alter gene-expression. However, approaches that allow to probe G4s in living cells without perturbing their folding dynamics are required to understand their biological roles in greater detail. Herein, we report a G4-specific fluorescent probe (SiR-PyPDS) that enables single-molecule and real-time detection of individual G4 structures in living cells. Live-cell single-molecule fluorescence imaging of G4s was carried out under conditions that use low concentrations of SiR-PyPDS (20 nM) to provide informative measurements representative of the population of G4s in living cells, without globally perturbing G4 formation and dynamics. Single-molecule fluorescence imaging and time-dependent chemical trapping of unfolded G4s in living cells, revealed that G4s fluctuate between folded and unfolded states. We also demonstrated that G4-formation in live cells is cell-cycle dependent and disrupted by chemical inhibition of transcription and replication. Our observations provide robust evidence in support of dynamic G4-formation in living cells.

Epigenetic upregulation of HOXC10 in non-small lung cancer cells.

The homeobox genes (HOX) have emerged as a new family of master regulators of development and cancer. In the current study, we examined the expression and function of HOXC10 in human non-small cell lung cancer (NSCLC). We observed increased expression of HOXC10 in the more aggressive human NSCLC cell line NCI-H23 over the well differentiated A549 cells. To elucidate the expression and function of HOXC10 in NSCLC cells, we employed RT-PCR, immunoblotting, methylation-specific PCR, apoptosis assays, and xenograft model. Overexpression of HOXC10 in A549 cells conveyed increased proliferation, reduced apoptosis, and accelerated tumor growth when transplanted into nude mice. In contrast, siRNA-mediated knockdown of HOXC10 in NCI-H23 cells reduced proliferation and increased apoptosis. Our results further indicated that hypomethylation of the CpG island in the HOXC10 promoter was critical to elevated expression of HOXC10 in NSCLC cells. Lastly, we identified a G-quadruplex in the HOXC10 promoter and its G-quadruplex formation was required for elevated expression of HOXC10 in NSCLC cells. Moreover our results suggest that disruption of G-quadruplex formation can silence HOXC10 expression in NSCLC cells. In summary, we report HOXC10 as a novel tumor promoting oncogene in NSCLC cells.

Contractions of the C-Terminal Domain of Saccharomyces cerevisiae Rpb1p Are Mediated by Rad5p.

The C-terminal domain (CTD) is an essential domain of the largest subunit of RNA polymerase II, Rpb1p, and is composed of 26 tandem repeats of a seven-amino acid sequence, YSPTSPS. Despite being an essential domain within an essential gene, we have previously demonstrated that the CTD coding region is genetically unstable. Furthermore, yeast with a truncated or mutated CTD sequence are capable of promoting spontaneous genetic expansion or contraction of this coding region to improve fitness. We investigated the mechanism by which the CTD contracts using a tet-off reporter system for RPB1 to monitor genetic instability within the CTD coding region. We report that contractions require the post-replication repair factor Rad5p but, unlike expansions, not the homologous recombination factors Rad51p and Rad52p. Sequence analysis of contraction events reveals that deleted regions are flanked by microhomologies. We also find that G-quadruplex forming sequences predicted by the QGRS Mapper are enriched on the noncoding strand of the CTD compared to the body of RPB1. Formation of G-quadruplexes in the CTD coding region could block the replication fork, necessitating post-replication repair. We propose that contractions of the CTD result when microhomologies misalign during Rad5p-dependent template switching via fork reversal.

Construction of Monomeric and Dimeric G-Quadruplex-Structured CpG Oligodeoxynucleotides for Enhanced Uptake and Activation in TLR9-Positive Macrophages.

The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5'-end [1668(5'-G5)], 3'-end [1668(3'-G5)], or within the sequence [1668(mid-G5)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5'-G5) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3'-G5). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3'-G5) decomposed more slowly in serum than 1668(5'-G5). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3'-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity.

Infrared Spectroscopy Reveals the Preferred Motif Size and Local Disorder in Parallel Stranded DNA G-Quadruplexes.

Infrared spectroscopy detects the formation of G-quadruplexes in guanine-rich nucleic acid sequences through shifts in the guanine C=O stretch mode. Here, we use ultrafast 2D infrared (IR) spectroscopy and isotope substitution to show that these shifts arise from vibrational delocalization among stacked G-quartets. This provides a direct measure of the sizes of locally ordered motifs in heterogeneous samples with substantial disordered regions. We find that parallel-stranded, potassium-bound DNA G-quadruplexes are limited to five consecutive G-quartets and 3-4 consecutive layers are preferred for longer polyguanine tracts. The resulting potassium-dependent G-quadruplex assembly landscape reflects the polyguanine tract lengths found in genomes, the ionic conditions prevalent in healthy mammalian cells, and the onset of structural disorder in disease states. Our study describes spectral markers that can be used to probe other G-quadruplex structures and provides insight into the fundamental limits of their formation in biological and artificial systems.

A dual-app nucleoside probe reports G-quadruplex formation and ligand binding in the long terminal repeat of HIV-1 proviral genome

We have developed a dual-app nucleoside analog, 5-selenophene-modified 2′-deoxyuridine (SedU), to probe the structure and ligand-binding properties of a G-rich segment present in the long terminal repeat (LTR) of the HIV-1 proviral DNA promoter region. The nucleoside probe is made of an environment-responsive fluorophore and X-ray crystallography phasing label (Se atom). SedU incorporated into LTR-IV sequence, fluorescently reports the formation of G-quadruplex (GQ) structure without affecting the native fold. Further, using the environment sensitivity of the probe, a fluorescence assay was designed to estimate the binding affinity of small molecule ligands to the GQ motif. An added feature of this probe system is that it would enable direct correlation of structure and recognition properties in solution and atomic level by using a combination of fluorescence and X-ray crystallography techniques.

Structure of two G-quadruplexes in equilibrium in the KRAS promoter.

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.

Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

Kinetics, conformation, stability, and targeting of G-quadruplexes from a physiological perspective

The particular enrichment of G-quadruplex-forming sequences near transcription start sites signifies the involvement of G-quadruplexes in the regulation of transcription. The characterization of G-quadruplex formation, which holds the key to understand the function it plays in physiological and pathological processes, is mostly performed under simplified in vitro experimental conditions. Formation of G-quadruplexes in cells, however, occurs in an environment far different from the ones in which the in vitro studies on G-quadruplexes are normally carried out. Therefore, the characteristics of G-quadruplex structures obtained under the in vitro conditions may not faithfully reveal how the G-quadruplexes would behave in a physiologically relevant situation. In this mini-review, we attempt to briefly summarize the differences in a few important characteristics, including kinetics, conformation, and stability of G-quadruplex formation observed under the two conditions to illustrate how the intracellular environment might affect the behavior of G-quadruplexes largely based on the previous work carried out in the authors’ laboratory. We also propose that unstable G-quadruplex variants may be better drug target candidates to improve selectivity and potency.

A Fluorescence Resonance Energy Transfer Probe Based on DNA-Modified Upconversion and Gold Nanoparticles for Detection of Lead Ions.

We report a new sensor for the specific detection of lead ions (Pb2+) in contaminated water based on fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) as donors and gold nanoparticles (Au NPs) as receptors. The UCNPs modified with Pb2+ aptamers could bind to Au NPs, which were functionalized with complementary DNA through hybridization. The green fluorescence of UCNPs was quenched to a maximum rate of 80% due to the close proximity between the energy donor and the acceptor. In the presence of Pb2+, the FRET process was broken because Pb2+ induced the formation of G-quadruplexes from aptamers, resulting in unwound DNA duplexes and separated acceptors from donors. The fluorescence of UCNPs was restored, and the relative intensity had a significant linear correlation with Pb2+ concentration from 0 to 50 nM. The sensor had a detection limit as low as 4.1 nM in a buffer solution. More importantly, the sensor exhibited specific detection of Pb2+ in complex metal ions, demonstrating high selectivity in practical application. The developed FRET prober may open up a new insight into the specific detection of environmental pollution.

Effects of Length and Loop Composition on Structural Diversity and Similarity of (G3TG3NmG3TG3) G-Quadruplexes.

A G-rich sequence containing three loops to connect four G-tracts with each ≥2 guanines can possibly form G-quadruplex structures. Given that all G-quadruplex structures comprise the stacking of G-quartets, the loop sequence plays a major role on their folding topology and thermal stability. Here circular dichroism, NMR, and PAGE are used to study the effect of loop length and base composition in the middle loop, and a single base difference in loop 1 and 3 on G-quadruplex formation of (G3HG3NmG3HG3) sequences with and without flanking nucleotides, where H is T, A, or C and N is T, A, C, or G. In addition, melting curve for G-quadruplex unfolding was used to provide relatively thermal stability of G-quadruplex structure after the addition of K+ overnight. We further studied the effects of K+ concentration on their stability and found structural changes in several sequences. Such (G3HG3NmG3HG3) configuration can be found in a number of native DNA sequences. The study of structural diversity and similarity from these sequences may allow us to establish the correlation between model sequences and native sequences. Moreover, several sequences upon interaction with a G-quadruplex ligand, BMVC, show similar spectral change, implying that structural similarity is crucial for drug development.

Human POT1 unfolds G-quadruplexes by conformational selection

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (−10.1 ± 0.3 kcal mol−1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.

K vs. Na Effects on the Self-Assembly of Guanosine 5'-Monophosphate: A Solution SAXS Structural Study+.

The hierarchical process of guanosine (G) self-assembly, leading in aqueous solution and in the presence of metal cations to the formation of G-quadruplexes, represents an intriguing topic both for the biological correlation with telomerase activity and for the nano-technological applications, as demonstrated by the current measured in a quadruplex wire 100 nm long. Similar to G-rich DNA sequences and G-oligonucleotides, the guanosine 5′-monophosphate (GMP) self-aggregates in water to form quadruplexes. However, due to the absence of a covalent axial backbone, this system can be very useful to understand the chemical-physical conditions that govern the guanosine supramolecular aggregation. We have then investigated by in-solution Synchrotron Small Angle X-ray Scattering technique the role of different cations in promoting the quadruplex formation as a function of concentration and temperature. Results show how potassium, with its peculiar biological traits, favours the G-quadruplex elongation process in respect to other cations (Na, but also NH and Li), determining the longest particles in solution. Moreover, the formation and the elongation of G-quadruplexes have been demonstrated to be controlled by both GMP concentration and excess cation content, even if they specifically contribute to these processes in different ways. The occurrence of condensed liquid crystalline phases was also detected, proving that excess cations play also unspecific effects on the effective charges on the G-quadruplex surface.

Elucidating Noncovalent Reaction Mechanisms: G-Quartet as an Intermediate in G-Quadruplex Assembly.

In analogy to covalent reactions, the understanding of noncovalent association pathways is fundamental to influence and control any supramolecular process. Following an approach that is reminiscent of covalent methodologies, we study here, for the first time, the mechanism of G-quadruplex formation in organic solvents. Our results support a reaction pathway in which the cation shifts the equilibrium towards a G-quartet transient intermediate, which then acts as a template in the formation of the G-quadruplex product.

Structural basis of G-quadruplex DNA recognition by the yeast telomeric protein Rap1.

G-quadruplexes are four-stranded nucleic acid structures involved in multiple cellular pathways including DNA replication and telomere maintenance. Such structures are formed by G-rich DNA sequences typified by telomeric DNA repeats. Whilst there is evidence for proteins that bind and regulate G-quadruplex formation, the molecular basis for this remains poorly understood. The budding yeast telomeric protein Rap1, originally identified as a transcriptional regulator functioning by recognizing double-stranded DNA binding sites, was one of the first proteins to be discovered to also bind and promote G-quadruplex formation in vitro. Here, we present the 2.4 Å resolution crystal structure of the Rap1 DNA-binding domain in complex with a G-quadruplex. Our structure not only provides a detailed insight into the structural basis for G-quadruplex recognition by a protein, but also gives a mechanistic understanding of how the same DNA-binding domain adapts to specifically recognize different DNA structures. The key observation is the DNA-recognition helix functions in a bimodal manner: In double-stranded DNA recognition one helix face makes electrostatic interactions with the major groove of DNA, whereas in G-quadruplex recognition a different helix face is used to make primarily hydrophobic interactions with the planar face of a G-tetrad.

Modulation of histone modifications and G-quadruplex structures by G-quadruplex-binding proteins

The functions of local conformations of non-B form DNA and RNA, such as the G-quadruplex, are thought to be regulated by their specific binding proteins. They regulate the formation of G-quadruplexes in cells and affect the biological functions of G-quadruplexes. Recent studies reported that G-quadruplexes regulate epigenetics through these G-quadruplex binding proteins. We discuss regulation of histone modifications through G-quadruplex RNA and its binding proteins which modulate the G-quadruplex conformations. G-quadruplex RNA is involved in telomere maintenance and transcription via histone modification. Furthermore, G-quadruplex binding proteins regulate formation and biological functions of G-quadruplexes through regulating their folding or unfolding. In this review, we will focus on the G-quadruplex binding proteins containing RRM and RGG domains.