Quadruplex Structures Research Articles

Dynamic alternative DNA structures in biology and disease.

Repetitive elements in the human genome, once considered 'junk DNA', are now known to adopt more than a dozen alternative (that is, non-B) DNA structures, such as self-annealed hairpins, left-handed Z-DNA, three-stranded triplexes (H-DNA) or four-stranded guanine quadruplex structures (G4 DNA). These dynamic conformations can act as functional genomic elements involved in DNA replication and transcription, chromatin organization and genome stability. In addition, recent studies have revealed a role for these alternative structures in triggering error-generating DNA repair processes, thereby actively enabling genome plasticity. As a driving force for genetic variation, non-B DNA structures thus contribute to both disease aetiology and evolution.

Assessing the Potential for DNA Quadruplex Formation in the Predatory Bacterium Bdellovibrio bacteriovorus.

During its life cycle, the predatory bacterium Bdellovibrio bacteriovorus switches between an attack and a growth phase, each of which is characterized by a distinct pattern of gene expression. Twenty-one potential G-quadruplex-forming sequences (PQFS) have been identified in the Bdellovibrio genome. These G-rich sequences are prevalent within open reading frames and nearly evenly distributed between the template and the coding strand, suggesting that they could play a role in gene expression and life cycle switching. Published transcriptomic data show that the genes nearest these sequences are not (de)activated together during the same phases of the life cycle. We explored the biophysical properties of three identified PQFS using circular dichroism (CD) spectroscopy and gel electrophoresis and demonstrated that all three sequences fold into stable unimolecular quadruplexes with distinct topologies. In the presence of their complementary strands, each forms an equilibrium mixture of duplex and quadruplex in which quadruplex formation is favored at higher temperatures. Once the quadruplexes are folded, they are slow to form a duplex when the complementary strand is added, with one sequence requiring the equivalent of many Bdellovibrio lifetimes to do so. Using a variety of cosolutes, we showed that molecular crowding mimicking cellular conditions stabilizes the quadruplex structures and induces structural transitions to the parallel topology regardless of the original topology. Taken together, these experiments suggest that Bdellovibrio PQFS are capable of forming quadruplexes in vivo and thereby playing a role in gene expression.

An all-deoxyribonucleic acid circuit for detection of human telomerase activity in solution and on paper

We report on the design of a simple all-DNA circuit with dual functions of signal amplification and signal reporting and its use for detection of human telomerase activity from cancer cells. The system utilizes a catalytic hairpin assembly (CHA) reaction for amplification, which produces split G-quadruplex outputs that assemble to form complete guanine quadruplex structures as reporting modules. As designed, a linear DNA sequence (the target) functions as a catalyst to drive cyclic programmed assembly of two hairpins, producing a DNA duplex with two guanine-rich sequences that assemble to form a complete Gq structure. The formation of the Gq element allows either fluorescence or colorimetric detection of the target. Examples are provided to demonstrate fluorescence detection of cancer cells’ telomerase activities in solution and the first example of a CHA-modulated colorimetric assay for detecting telomerase activities of cancer cells using a simple paper device.

Non-G Base Tetrads.

Tetrads (or quartets) are arrangements of four nucleobases commonly involved in the stability of four-stranded nucleic acids structures. Four-stranded or quadruplex structures have attracted enormous attention in the last few years, being the most extensively studied guanine quadruplex (G-quadruplex). Consequently, the G-tetrad is the most common and well-known tetrad. However, this is not the only possible arrangement of four nucleobases. A number of tetrads formed by the different nucleobases have been observed in experimental structures. In most cases, these tetrads occur in the context of G-quadruplex structures, either inserted between G-quartets, or as capping elements at the sides of the G-quadruplex core. In other cases, however, non-G tetrads are found in more unusual four stranded structures, such as i-motifs, or different types of peculiar fold-back structures. In this report, we review the diversity of these non-canonical tetrads, and the structural context in which they have been found.

Polymerase η Recruits DHX9 Helicase to Promote Replication across Guanine Quadruplex Structures.

DNA polymerase η (Pol η) catalyzes accurate bypass of ultraviolet light-induced cyclobutane pyrimidine dimers, and it also functions in several other related processes, including bypassing DNA with unusual structures. Here, we performed unbiased proteome-wide profiling of Pol η-interacting proteins by using two independent approaches, i.e., proximity labeling and affinity pull-down followed by LC-MS/MS analysis. We identified several helicases, including DHX9, as novel Pol η-interacting proteins. Additionally, ChIP-Seq analysis showed that Pol η is enriched at guanine quadruplex (G4) structure sites in chromatin. Moreover, Pol η promotes the recruitment of DHX9 to G4 structure loci in chromatin and facilitates DHX9-mediated unwinding of G4 structures. Deficiency in Pol η or DHX9 leads to attenuated replication across G4 regions in genomic DNA. Together, we unveiled the interaction between Pol η and DHX9 and demonstrated that the interaction promotes the replicative bypass of G4 structures in chromatin.

G-Quadruplex selectivity and cytotoxicity of a guanidine-encapsulated porphyrin-cyclodextrin conjugate

G-Quadruplex DNAs represent out-of-the-way nucleic acid conformations, frequently formed by guanine-rich sequences. They have emanated as cancer-associated targets for designed small molecules. The variation in the binding affinity of the synthesized compounds to duplex and quadruplex structures is an intriguing quest, solved by spectroscopic analysis. In this paper, we report the synthesis of a porphyrin-cyclodextrin conjugate, characterized by utilizing FT-IR, NMR, and mass spectrometry. Further, two benzimidazolylguanidines are synthesized which form host: guest complexes with the porphyrin-cyclodextrin conjugate. The structure of the complexes is optimized by analyzing their 2D ROESY spectra. The interactions of the host, guest, and the host: guest complexes with the duplex (calf thymus DNA) and quadruplex (kit22) nucleic acids are investigated employing UV–vis, fluorescence, circular dichroism, and DNA melting experiments. The calculated strengths of the compounds' binding with kit22 are in the order of 106, which is larger than those observed for the duplex DNA binding. The significant G-quadruplex selectivity of the host: guest complex of anthracenyl-benzimidazolylguanidine is discussed in detail. Further, the in vitro cytotoxicity of the compounds on MCF-7 cell lines is examined. The host: guest complexes show enhanced half-maximal inhibitory concentration values compared to the un-complexed compounds.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Dissecting G-Quadruplexes and I-Motifs in Cis-Regulatory Regions of Mammals and Plants

The evolutionary constraints of a gene consist of cis-gene regulatory regions, such as promoters and enhancers, which contribute to the regulation of gene expression. Several genomic and computational studies stated the roles of G-quadruplex and i-motif structures in vital cellular processes like transcription, translation, gene regulation, etc. The formation of these non-B DNA structures is supported by the occurrence of unique repeated sequences. However, many studies lean toward understanding the role of the G-quadruplex, and only recent studies indicated i-motif significance. In this study, we attempted to dissect the enrichment of G-quadruplex and i-motifs in promoter regions of mammals and plants. To this end, we employed the genomic sequences encompassing -500 to +500 region relative to the gene start positions in mammals and plants retrieved from the UCSC browser and Plant Genome database (PlantGDB). We computed the putative G-quadruplexes and i-motifs with well-recognized regular expression sequence patterns. We observed that G-quadruplex motifs showed preponderance in mammals, algal species, namely, green algae, and Chlamydomonas when compared to plants. Contrastingly i-motifs are enriched in both monocot and dicot plants compared to G-quadruplex motifs. The comparative examinations in this study revamp our understanding of the two quadruplex structures and their emerging functional roles in complex eukaryotes.

Telomere Length Regulation.

The number of (TTAGGG)n repeats at the ends of chromosomes is highly variable between individual chromosomes, between different cells and between species. Progressive loss of telomere repeats limits the proliferation of pre-malignant human cells but also contributes to aging by inducing apoptosis and senescence in normal cells. Despite enormous progress in understanding distinct pathways that result in loss and gain of telomeric DNA in different cell types, many questions remain. Further studies are needed to delineate the role of damage to telomeric DNA, replication errors, chromatin structure, liquid-liquid phase transition, telomeric transcripts (TERRA) and secondary DNA structures such as guanine quadruplex structures, R-loops and T-loops in inducing gains and losses of telomere repeats in different cell types. Limitations of current telomere length measurements techniques and differences in telomere biology between species and different cell types complicate generalizations about the role of telomeres in aging and cancer. Here some of the factors regulating the telomere length in embryonic and adult cells in mammals are discussed from a mechanistic and evolutionary perspective.

Novel L-RNA Aptamer Controls APP Gene Expression in Cells by Targeting RNA G-Quadruplex Structure.

Guanine quadruplex (G4) structure is a four-stranded nucleic acid secondary structure motif with unique chemical properties and important biological roles. Amyloid precursor protein (APP) is an Alzheimer's disease (AD)-related gene, and recently, we reported the formation of RNA G4 (rG4) at the 3'UTR of APP mRNA and demonstrated its repressive role in translation. Herein, we apply rG4-SELEX to develop a novel L-RNA aptamer, L-Apt.8f, which binds to APP 3'UTR D-rG4 strongly with subnanomolar affinity. We structurally characterize the aptamer and find that it contains a thermostable and parallel G4 motif, and mutagenesis analysis identifies the key nucleotides that are involved in the target recognition. We also reveal that the L-Apt.8f-APP D-rG4 interaction is enantiomeric-, magnesium ion-, and potassium ion-dependent. Notably, L-Apt.8f preferentially recognizes APP rG4 over other structural motifs, and it can control the APP reporter gene and native transcript translation in cells. Our work introduces a novel strategy and reports a new L-aptamer candidate to target APP 3'UTR rG4 structure, which laid the foundation for further applying L-RNA as an important class of biomolecule for practical L-aptamer-based targeting and controlling of gene expression in cells.

Drug discovery of small molecules targeting the higher-order hTERT promoter G-quadruplex.

DNA G-quadruplexes (G4s) are now widely accepted as viable targets in the pursuit of anticancer therapeutics. To date, few small molecules have been identified that exhibit selectivity for G4s over alternative forms of DNA, such as the ubiquitous duplex. We posit that the lack of current ligand specificity arises for multiple reasons: G4 atomic models are often small, monomeric, single quadruplex structures with few or no druggable pockets; targeting G-tetrad faces frequently results in the enrichment of extended electron-deficient polyaromatic end-pasting scaffolds; and virtual drug discovery efforts often under-sample chemical search space. We show that by addressing these issues we can enrich for non-standard molecular templates that exhibit high selectivity towards G4s over other forms of DNA. We performed an extensive virtual screen against the higher-order hTERT core promoter G4 that we have previously characterized, targeting 12 of its unique loop and groove pockets using libraries containing 40 million drug-like compounds for each screen. Using our drug discovery funnel approach, which utilizes high-throughput fluorescence thermal shift assay (FTSA) screens, microscale thermophoresis (MST), and orthogonal biophysical methods, we have identified multiple unique G4 binding scaffolds. We subsequently used two rounds of catalogue-based SAR to increase the affinity of a disubstituted 2-aminoethyl-quinazoline that stabilizes the higher-order hTERT G-quadruplex by binding across its G4 junctional sites. We show selectivity of its binding affinity towards hTERT is virtually unaffected in the presence of near-physiological levels of duplex DNA, and that this molecule downregulates hTERT transcription in breast cancer cells.

DrawTetrado to create layer diagrams of G4 structures.

MotivationQuadruplexes are specific 3D structures found in nucleic acids. Due to the exceptional properties of these motifs, their exploration with the general-purpose bioinformatics methods can be problematic or insufficient. The same applies to visualizing their structure. A hand-drawn layer diagram is the most common way to represent the quadruplex anatomy. No molecular visualization software generates such a structural model based on atomic coordinates.ResultsDrawTetrado is an open-source Python program for automated visualization targeting the structures of quadruplexes and G4-helices. It generates static layer diagrams that represent structural data in a pseudo-3D perspective. The possibility to set color schemes, nucleotide labels, inter-element distances or angle of view allows for easy customization of the output drawing.Availability and implementationThe program is available under the MIT license at https://github.com/RNApolis/drawtetrado.

Human Virus Genomes Are Enriched in Conserved Adenine/Thymine/Uracil Multiple Tracts That Pause Polymerase Progression

The DNA secondary structures that deviate from the classic Watson and Crick base pairing are increasingly being reported to form transiently in the cell and regulate specific cellular mechanisms. Human viruses are cell parasites that have evolved mechanisms shared with the host cell to support their own replication and spreading. Contrary to human host cells, viruses display a diverse array of nucleic acid types, which include DNA or RNA in single-stranded or double-stranded conformations. This heterogeneity improves the possible occurrence of non-canonical nucleic acid structures. We have previously shown that human virus genomes are enriched in G-rich sequences that fold in four-stranded nucleic acid secondary structures, the G-quadruplexes.Here, by extensive bioinformatics analysis on all available genomes, we showed that human viruses are enriched in highly conserved multiple A (and T or U) tracts, with such an array that they could in principle form quadruplex structures. By circular dichroism, NMR, and Taq polymerase stop assays, we proved that, while A/T/U-quadruplexes do not form, these tracts still display biological significance, as they invariably trigger polymerase pausing within two bases from the A/T/U tract. “A” bases display the strongest effect. Most of the identified A-tracts are in the coding strand, both at the DNA and RNA levels, suggesting their possible relevance during viral translation. This study expands on the presence and mechanism of nucleic acid secondary structures in human viruses and provides a new direction for antiviral research.

1H-imidazo[4,5-f][1,10]phenanthroline carbohydrate conjugates: Synthesis, DNA interactions and cytotoxic activity

Here we present a novel family of carbohydrate conjugates based on the 2-aryl-imidazo[4,5-f][1,10]phenanthroline core modified with carbohydrates (carb-APIPs). The hybrid compounds were prepared by direct treatment of the unprotected carbohydrate with 2-(4-aminophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (APIP). The N-glycosylation reactions with the monosaccharides tested afforded stereoselectively the more stable N-β-glycopyranosylamines that, in solution, underwent a dynamic equilibrium leading to anomeric mixtures with a small participation of the α isomer. DNA interaction experiments with telomeric G-quadruplex DNA included DNA FRET melting assays, circular dichroism, and equilibrium dialysis and revealed that the novel carb-APIPs bind the G-quadruplex structure with high affinity. Interestingly, the presence of the carbohydrate confers good selectivity towards the telomeric quadruplex structure, as suggested by competition DNA FRET melting assays. Besides the extended aromatic surface that allows π-stacking interactions, the carbohydrate part of the conjugate may contribute to groove binding recognition, as indicated by viscosity experiments. In addition, the novel carb-APIPs showed significant cytotoxic properties in PC3 and HeLa cells and, to a lesser extent, in MCF7 cells and normal human fibroblasts (HFF1).

Long promoter sequences form higher-order G-quadruplexes: an integrative structural biology study of c-Myc, k-Rasand c-Kit promoter sequences.

We report on higher-order G-quadruplex structures adopted by long promoter sequences obtained by an iterative integrated structural biology approach. Our approach uses quantitative biophysical tools (analytical ultracentrifugation, small-angle X-ray scattering, and circular dichroism spectroscopy) combined with modeling and molecular dynamics simulations, to derive self-consistent structural models. The formal resolution of our approach is 18 angstroms, but in some cases structural features of only a few nucleotides can be discerned. We report here five structures of long (34–70 nt) wild-type sequences selected from three cancer-related promoters: c-Myc, c-Kit and k-Ras. Each sequence studied has a unique structure. Three sequences form structures with two contiguous, stacked, G-quadruplex units. One longer sequence from c-Myc forms a structure with three contiguous stacked quadruplexes. A longer c-Kit sequence forms a quadruplex-hairpin structure. Each structure exhibits interfacial regions between stacked quadruplexes or novel loop geometries that are possible druggable targets. We also report methodological advances in our integrated structural biology approach, which now includes quantitative CD for counting stacked G-tetrads, DNaseI cleavage for hairpin detection and SAXS model refinement. Our results suggest that higher-order quadruplex assemblies may be a common feature within the genome, rather than simple single quadruplex structures.

Acid-Resistant and Physiological pH-Responsive DNA Hydrogel Composed of A-Motif and i-Motif toward Oral Insulin Delivery.

An acid-resistant DNA hydrogel that is stable in an extremely acidic environment with pH as low as 1.2 has not been reported before, largely due to the instability of DNA-hybridized structures. To achieve this, adenine (A)-rich and cytosine (C)-rich oligonucleotides are rationally designed and integrated to form copolymers with acrylamide monomers via free-radical polymerization. In an acidic environment (pH 1.2-6.0), the generated copolymers form a hydrogel state, which is cross-linked by parallel A-motif duplex configurations (pH 1.2-3.0) and quadruplex i-motif structures (pH 4.0-6.0) due to the protonation of A and C bases, respectively. Specifically, the protonated A-rich sequences under pH 1.2-3.0 form a stable parallel A-motif duplex cross-linking unit through reverse Hoogsteen interaction and electrostatic attraction. Hemi-protonated C bases under mildly acidic pH (4.0-6.0) form quadruplex i-motif cross-linking configuration via Hoogsteen interaction. Under physiological pH, both A and C bases deprotonated, resulting in the separation of A-motif and i-motif to A-rich and C-rich single strands, respectively, and thereby the dissociation of the DNA hydrogel into the solution state. The acid-resistant and physiological pH-responsive DNA hydrogel was further developed for oral drug delivery to the hostile acidic environment in the stomach (pH 1.2), duodenum (pH 5.0), and small intestine (pH 7.2), where the drug would be released and absorbed. As a proof of concept, insulin was encapsulated in the DNA hydrogel and orally administered to diabetic rats. In vitro and in vivo studies demonstrated the potential usage of the DNA hydrogel for oral drug delivery.

Genomic landscape, polymorphism and possible LINE-associated delivery of G-quadruplex motifs in the bovine genes.

G-Quadruplex structures are non-B DNA structures that occur in regions carrying short runs of guanines. They are implicated in several biological processes including transcription, translation, replication and telomere maintenance as well as in several pathological conditions like cancer and thus have gained the attention of the scientific community. The rise of the -omics era significantly affected the G-quadruplex research and the genome-wide characterization of G-Quadruplexes has been rendered a necessary first step towards applying genomics approaches for their study. While in human and several model organisms there is a considerable number of works studying genome-wide the DNA motifs with potential to form G-quadruplexes (G4-motifs), there is a total absence of any similar studies regarding livestock animals. The objectives of the present study were to provide a detailed characterization of the bovine genic G4-motifs' distribution and properties and to suggest a possible mechanism for the delivery of G4-motifs in the genes. Our data indicate that the distribution of G4-motifs within bovine genes and the annotation of said genes to Gene Ontology terms are similar to what is already shown for other organisms. By investigating their structural characteristics and polymorphism, it is obvious that the overall stability of the putative quadruplex structures is in line with the current notion in the G4 field. Similarly to human, the bovine G4-motifs are overrepresented in specific LINE repeat elements, the L1_BTs in the case of cattle. We highlight the potential role of these elements as vehicles for delivery of G4-motifs in the introns of the bovine genes. Lastly, it seems that a basis exists for connecting traits of agricultural importance to the genetic variation of G4-motifs, thus, the value of cattle as an interesting new model organism for G4-related genetic studies might be worth investigating.

P46 Antibiotic susceptibility response of adeABC in the presence of a G-quadruplex DNA molecule

BackgroundG-quadruplexes are secondary nucleotide structures formed from hydrogen binding and stacking of guanine bases at their Watson–Crick and Hoogsteen edges. These structures have confirmed roles in specific genes such as within replication and switching on or off transcription and translation. However, the full potential of these molecular tools has yet to be fully unlocked. The utilization of quadruplexes as molecular tools as well as the further understanding of their mechanistic roles within genetic regulation could lead to the development of novel therapeutic strategies against drug resistance.MethodsAntibiotic susceptibility profiling studies in 43 clinical Acinetobacter baumannii strains revealed MDR properties to ertapenem, meropenem, tetracycline and ciprofloxacin. The role of the efflux pump genes, adeABC, to the resistance observed was further confirmed by an inhibitory assay in the presence of PaβN (30 mg/L). Results showed a 97.8% decrease in bacterial growth with ertapenem at a concentration of 125 mg/L when compared with cells without PaβN. However, PaβN is not a viable pharmaceutical compound, as it has toxic properties harmful to life. Therefore, quadruplexes having similar function of inhibiting the mechanism of efflux genes in resistance introduces a substantial alternative in developing a novel antimicrobial therapy. Potential quadruplex forming sequences within the adeABC gene of A. baumannii (NZ_CP009257.1) using a predictive software tool, QGRS Mapper, were identified. Assembly of the identified sequences was initiated through the instruction of monovalent metal cations (potassium or sodium; 6.8 pH) by heat denaturation assays. Assembly products were further analysed using UV-Vis spectrophotometry, where a thermal difference spectrum (95°C–20°C) was produced, allowing the observation of characteristic quadruplex spectra. A second technique, DNase Assay, was used for additional confirmation of quadruplex assembly, as quadruplexes are resistant to the enzyme's degradation activity.ResultsFive assembled sequences indicated results characteristic of a quadruplex structure formation. A sequence, ade9, was then introduced at 1 AU concentration (260 nm) into the bacterial cells of NCTC 12156 strain and incubated with antibiotics at 37°C. It is expected that the introduced assembled quadruplexes would competitively bind to quadruplex-associated-proteins innate within the cells. Where a quadruplex present in cell results in the upregulation of a gene, the introduced quadruples should be observed to result in down-regulation of the efflux pump, increasing susceptibility to the antimicrobial agents. Experiments showed results accepting the proposed hypothesis, with a significant decrease in bacteria growth, indicating a decrease in resistance to the antibiotics.ConclusionsResistance of microbes to available therapeutics continues to gain an increased level of concern, posing a threat to life and economy. Identification of the roles of quadruplexes in antimicrobial genes has the potential to unlock a new era for antibiotic therapeutics. Targeting the action of these physiologically active structures may inhibit resistance mechanisms and restore the efficacy of many antimicrobial chemotherapies.

G quadruplex structures in NEAT1: potential functions in regulating its stability and its interactions with the fused in sarcoma protein

Paraspeckles are membraneless organelles found within mammalian nuclei comprised of RNA and proteins. The long non-coding RNA, nuclear enriched abundant transcript 1 (NEAT1) has been shown to be essential for paraspeckles formation. NEAT1 has two isoforms, a short one of ∼3.7 kB (NEAT1_1) and a long one of ∼22.7 kB (NEAT1_2), and each paraspeckle may contain up to fifty NEAT1 RNAs. We and others identified five regions within NEAT1 that have the potential to form G-quadruplex structures (GQs), four of which are common to both isoforms and one found only in the long isoform NEAT1_2.

Computational and Experimental Characterization of rDNA and rRNA G-Quadruplexes.

DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. G-quadruplexes have been unambiguously shown to exist both in vitro and in vivo, including in the guanine (G)-rich DNA genes encoding pre-ribosomal RNA (pre-rRNA), which is transcribed in the cell's nucleolus. Recent studies strongly suggest that these DNA sequences ("rDNA"), and the transcribed rRNA, are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis, but the structures of ribosomal G-quadruplexes at atomic resolution are unknown and very little biophysical characterization has been performed on them to date. In the present study, circular dichroism (CD) spectroscopy is used to show that two putative rDNA G-quadruplex sequences, NUC 19P and NUC 23P and their counterpart rRNAs, predominantly adopt parallel topologies, reminiscent of the analogous telomeric quadruplex structures. Based on this information, we modeled parallel topology atomistic structures of the putative ribosomal G-quadruplexes. We then validated and refined the modeled ribosomal G-quadruplex structures using all-atom molecular dynamics (MD) simulations with the CHARMM36 force field in the presence and absence of stabilizing K+. Motivated by preliminary MD simulations of the telomeric parallel G-quadruplex (TEL 24P) in which the K+ ion is expelled, we used updated CHARMM36 force field K+ parameters that were optimized, targeting the data from quantum mechanical calculations and the polarizable Drude model force field. In subsequent MD simulations with optimized CHARMM36 parameters, the K+ ions are predominantly in the G-quadruplex channel and the rDNA G-quadruplexes have more well-defined, predominantly parallel-topology structures as compared to rRNA. In addition, NUC 19P is more structured than NUC 23P, which contains extended loops. Results from this study set the structural foundation for understanding G-quadruplex functions and the design of novel chemotherapeutics against these nucleolar targets and can be readily extended to other DNA and RNA G-quadruplexes.