G-quadruplex Formation Research Articles

A 1,10-phenanthroline derivative selectively targeting telomeric G-quadruplex induces cytoprotective autophagy, causing apoptosis of gastric cancer cells

AimsThis study aimed to evaluate the ability of compound 13d to induce autophagy and to promote apoptosis of tumor cells and its interaction mechanism. Materials and methodsUsing CCK-8 assay, transwell assay, fluorescence resonance energy transfer melting analysis (FRET), transmission electron microscopy, flow cytometry assay, immunofluorescence assay, Western blot analysis, and wound healing assay. Key findingsThe results indicated that compound 13d could induce autophagy and apoptosis of gastric cancer cells. Moreover, the findings of CCK-8 assay, colony formation, migration and invasion assay, and wound healing assay revealed that compound 13d would effectively inhibit cell proliferation, migration, and invasion. Its IC50 value is about 2.4 μM against gastric cancer cells, which is similar to positive drug‑platinum. 13d specific induction of telomere G-quadruplex formation was proved in extracellular FRET melting assay, and indirectly affected telomerase activity. G-quadruplex formation promoted cell apoptosis and autophagy. Upon incorporating the autophagy inhibitors 3-MA and HCQ, the expression of the autophagy marker protein LC3 was then checked, suggesting that the compound 13d influences the autophagy flux. Furthermore, knocking down the autophagy-related gene Atg5 to reduce the level of autophagy enhances the anti-tumor activity and increases apoptotic cells' proportion. Mechanistic experiments have shown that blocking the Akt/m-TOR signal pathway plays a crucial role in autophagy and G-quadruplex induced telomere dysfunction. DNA damage is the leading cause of autophagy. Compound 13d combined with autophagy inhibitor can inhibit tumor cells more effectively. SignificanceOur findings demonstrate that compound 13d as a telomeric G-quadruplex ligand induces Telomere dysfunction, DNA damage response, autophagy, and apoptosis in gastric cancer cells by blocking the Akt/m-TOR signaling pathway.

An ultra-high-sensitivity electrochemiluminescence aptasensor for Pb2+ detection based on the synergistic signal-amplification strategy of quencher abscission and G-quadruplex generation

Signal amplification provides an effective way to improve detection performance. Herein, an ultrasensitive electrochemiluminescence (ECL) aptasensor for Pb2+ detection was developed based on a dual signal-amplification strategy of the abscission of a quencher and the generation of a G-quadruplex by one-step and simultaneous way. Nitrogen-doped carbon quantum dots linked with complementary DNA (cDNA-NCQDs) at the sensing interface was applied as the quencher of a tris(4,4′-dicarboxylic acid-2,2′-bipyridyl)ruthenium(II) (Ru(dcbpy)32+)/tripropylamine system to minimize the ECL signal due to the intermolecular hydrogen bond-induced energy-transfer process. Upon the addition of Pb2+, its specific binding with the aptamer triggered the abscission of cDNA-NCQDs, accompanied by the formation of G-quadruplex on the surface of the electrode, both of which amplified the intensity of the light emission. The ECL amplification efficiency induced by the above two mechanisms (78.6%) was valuably greater than that of their sum value (69.3%). This synergistic effect resulted in high detection sensitivity of the ECL aptasensor, which allowed to thereby obtain Pb2+ measurements in the range of 1 fM - 10 nM with an ultra-low detection limit of 0.19 fM. The Pb2+-mediated synergistic signal-amplification ECL strategy can provide a new approach for integrating various amplification strategies.

Transliteration of synthetic genetic enzymes.

Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration—a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2′-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding.

Wilms’ tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences

Wilms’ tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.

Thioflavin T fluorescence and NMR spectroscopy suggesting a non-G-quadruplex structure for a sodium binding aptamer embedded in DNAzymes

Recently, a Na+-binding aptamer was reported to be embedded in a few RNA-cleaving DNAzymes, including NaA43, Ce13d, and NaH1. The Na+ aptamer consists of multiple GG stretches, which is a prerequisite for the formation of G-quadruplex (G4) structures. These DNAzymes require Na+ for activity but show no activity in the presence of K+ or other metal ions. Given that DNA can selectively bind K+ by forming a G4 structure, this work aims to answer whether this Na+ aptamer also uses a G4 to bind Na+. Through comparative ThT fluorescence spectrometry studies, while a control G4 DNA exhibited notable fluorescence enhancement up to 5 mM K+ with a Kd of 0.28 ± 0.06 mM, the Ce13d DNAzyme fluorescence was negligibly perturbed with similar concentrations of K+. Opposed to this, Ce13d displayed specific remarkable fluorescence decrease with low millimolar concentrations of Na+. NMR experiments at two different pH values suggest that Ce13d adopts a significantly different conformation or equilibrium of conformations in the presence of Na+ versus K+ and has a more stable structure in the presence of Na+. Additionally, absence of characteristic G4 peaks in one-dimensional 1H NMR suggest that G4 is not responsible for the Na+ binding. This hypothesis is confirmed by the absence of characteristic peaks in the CD spectra of this sequence. Therefore, we concluded that the aptamer must be selective for Na+ and that it binds Na+ using a structural element that does not contain G4.

Combining Electrospray Mass Spectrometry (ESI-MS) and Computational Techniques in the Assessment of G-Quadruplex Ligands: A Hybrid Approach to Optimize Hit Discovery.

Guanine-rich sequences forming G-quadruplexes (GQs) are present in several genomes, ranging from viral to human. Given their peculiar localization, the induction of GQ formation or GQ stabilization with small molecules represents a strategy for interfering with crucial biological functions. Investigating the recognition event at the molecular level, with the aim of fully understanding the triggered pharmacological effects, is challenging. Native electrospray ionization mass spectrometry (ESI-MS) is being optimized to study these noncovalent assemblies. Quantitative parameters retrieved from ESI-MS studies, such as binding affinity, the equilibrium binding constant, and sequence selectivity, will be overviewed. Computational experiments supporting the ESI-MS investigation and boosting its efficiency in the search for GQ ligands will also be discussed with practical examples. The combination of ESI-MS and in silico techniques in a hybrid high-throughput-screening workflow represents a valuable tool for the medicinal chemist, providing data on the quantitative and structural aspects of ligand–GQ interactions.

Two coexisting pseudo-mirror heteromolecular telomeric G-quadruplexes in opposite loop progressions differentially recognized by a low equivalent of Thioflavin T.

The final 3′-terminal residue of the telomeric DNA G-overhang is inherently less precise. Here, we describe how alteration of the last 3′-terminal base affects the mutual recognition between two different G-rich oligomers of human telomeric DNA in the formation of heteromolecular G-quadruplexes (hetero-GQs). Associations between three- and single-repeat fragments of human telomeric DNA, target d(GGGTTAGGGTTAGGG) and probe d(TAGGGT), in Na+ solution yield two coexisting forms of (3 + 1) hybrid hetero-GQs: the kinetically favourable LLP-form (left loop progression) and the thermodynamically controlled RLP-form (right loop progression). However, only the adoption of a single LLP-form has been previously reported between the same probe d(TAGGGT) and a target variant d(GGGTTAGGGTTAGGGT) having one extra 3′-end thymine. Moreover, the flanking base alterations of short G-rich probe variants also significantly affect the loop progressions of hetero-GQs. Although seemingly two pseudo-mirror counter partners, the RLP-form exhibits a preference over the LLP-form to be recognized by a low equivalent of fluorescence dye thioflavin T (ThT). To a greater extent, ThT preferentially binds to RLP hetero-GQ than with the corresponding telomeric DNA duplex context or several other representative unimolecular GQs.

Impact of a Single Nucleotide Change or Non-Nucleoside Modifications in G-Rich Region on the Quadruplex-Duplex Hybrid Formation.

In this paper, a method to discriminate between two target RNA sequences that differ by one nucleotide only is presented. The method relies on the formation of alternative structures, i.e., quadruplex–duplex hybrid (QDH) and duplex with dangling ends (Dss), after hybridization of DNA or RNA G-rich oligonucleotides with target sequences containing 5′–GGGCUGG–3′ or 5′–GGGCGGG–3′ fragments. Using biophysical methods, we studied the effect of oligonucleotide types (DNA, RNA), non-nucleotide modifications (aliphatic linkers or abasic), and covalently attached G4 ligand on the ability of G-rich oligonucleotides to assemble a G-quadruplex motif. We demonstrated that all examined non-nucleotide modifications could mimic the external loops in the G-quadruplex domain of QDH structures without affecting their stability. Additionally, some modifications, in particular the presence of two abasic residues in the G-rich oligonucleotide, can induce the formation of non-canonical QDH instead of the Dss structure upon hybridization to a target sequence containing the GGGCUGG motif. Our results offer new insight into the sequential requirements for the formation of G-quadruplexes and provide important data on the effects of non-nucleotide modifications on G-quadruplex formation.

An analytical study of lipid-oligonucleotide aggregation properties

Lipid-oligonucleotides (LON) attract great interest as supramolecular scaffolds to improve the intracellular delivery of nucleic acids. Analytical characterization of LON assemblies is critical to formulation development, understanding in-vivo performance, as well as quality control. For this study, we selected LONs featuring different modifications on both oligonucleotide (with or without a G4 prone sequence) and lipid (mono or bis-alkyl chain covalently attached to the oligonucleotide sequence). Size exclusion chromatography (SEC) and, for the first time, capillary electrophoresis (CE) were investigated to study LON supramolecular self-assemblies. Results were correlated to those obtained with conventional physico-chemical characterization techniques i.e. gel electrophoresis, dynamic light scattering, and circular dichroism. In SEC, a separation between LON monomers and micelles was achieved in 5min on a TSK-gel G3000PW column at 70°C with 100% water, as mobile phase. CE conditions were optimized using a fused-silica capillary length of 10.0cm effective length at 15°C. Different background electrolytes were tested by varying the nature and the concentration of salts added. A sodium tetraborate buffer with 75mM NaCl appeared suitable to promote LON assembly. CE offers benefits to LON micelle analysis in terms of speed of analysis, high resolution, and low quantity of sample injected. Moreover, CE provides an appropriate tool to assess the impact of media of biological relevance on LON self-assembly. In this work, the key role of lipophilic tails and the formation of tetramolecular G-quadruplexes on the stability of LON micelles was confirmed.

The role of G-Quadruplex DNA in Paraspeckle formation in cancer

Paraspeckles are RNA–protein structures within the nucleus of mammalian cells, capable of orchestrating various biochemical processes. An overexpression of the architectural component of paraspeckles, a long non-coding RNA called NEAT1 (Nuclear Enriched Abundant Transcript 1), has been linked to a variety of cancers and is often associated with poor patient prognosis. Thus, there is an accumulating interest in the role of paraspeckles in carcinogenesis, however there is a limited understanding of how NEAT1 expression is regulated. Here, we demonstrate that both nuclear G-quadruplex (G4) and paraspeckle formation are significantly increased in a human breast cancer cell line compared to non-tumorigenic breast cells. Moreover, we identified and characterized G4-forming sequences within the NEAT1 promoter and demonstrate stabilization of G4 DNA with a G4-stabilizing small molecule results in a significant alteration in both paraspeckle formation and NEAT1 expression. This G4-mediated alteration of NEAT1 at both the transcriptional and post-transcriptional levels was evident in U2OS osteosarcoma cells, MCF-7 breast adenocarcinoma and MDA-MB-231 triple negative breast cancer cells.

The different biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of osteosarcoma are attributed to G-quadruplex.

ObjectiveOsteosarcoma (OS) is characterized by high levels of the tumour‐associated inflammatory microenvironment. Moreover, in approximately 60% of OS, telomere length is maintained by alternative lengthening of telomeres (ALT) pathway. Whether the ALT pathway can be exploited for OS therapeutic treatment and how the OS inflammatory microenvironment influences the anti‐cancer drug effect remains unknown. Here, we examined the biological effects of TMPyP4 and cisplatin in the inflammatory microenvironment of OS cells.Materials and methodsImmunofluorescence in situ hybridization (IF‐FISH) and C‐circle experiments were used to detect the G‐quadruplex and ALT activity. The redox potential of single guanine, G‐quadruplex and G‐quadruplex/TMPyP4 was evaluated by the lowest unoccupied molecular orbital energy (LUMO), zeta potential and cyclic voltammetry. Cell viability, flow cytometry and apoptosis, Western blot, comet assay, adhesion, transwell and scratch experiments were performed to compare the anti‐tumour proliferation and migration effects of TMPyP4 and cisplatin in the inflammatory microenvironment.ResultsThis study indicated that compared with cisplatin, TMPyP4 could induce the formation of human telomeres and FAK G‐quadruplex in vitro and in vivo, and TMPyP4‐treated OS cells showed fewer extrachromosomal C‐circles and fewer ALT‐associated promyelocytic leukaemia bodies. Consequently, the ALT activity and FAK‐related cell migration were suppressed by TMPyP4. Mechanistically, the formation of G‐quadruplex resulted in both lower redox potential than G within the genome and FAK transcription inhibition, and TMPyP4 could enhance this phenomenon, especially in the inflammatory microenvironment.ConclusionsOur results reveal that TMPyP4 is more suitable for OS treatment than cisplatin.

Characterization of G4 DNA formation in mitochondrial DNA and their potential role in mitochondrial genome instability.

Mitochondria possess their own genome which can be replicated independently of nuclear DNA. Mitochondria being the powerhouse of the cell produce reactive oxygen species, due to which the mitochondrial genome is frequently exposed to oxidative damage. Previous studies have demonstrated an association of mitochondrial deletions to aging and human disorders. Many of these deletions were present adjacent to non-B DNA structures. Thus, we investigate noncanonical structures associated with instability in mitochondrial genome. Insilico studies revealed the presence of >100 G-quadruplex motifs (of which 5 have the potential to form 3-plate G4 DNA), 23 inverted repeats, and 3 mirror repeats in the mitochondrial DNA (mtDNA). Further analysis revealed that among the deletion breakpoints from patients with mitochondrial disorders, majority are located at G4 DNA motifs. Interestingly, ~50% of the deletions were at base-pair positions 8271-8281, ~35% were due to deletion at 12362-12384, and ~12% due to deletion at 15516-15545. Formation of 3-plate G-quadruplex DNA structures at mitochondrial fragile regions was characterized using electromobility shift assay, circular dichroism (CD), and Taq polymerase stop assay. All 5 regions could fold into both intramolecular and intermolecular G-quadruplex structures in a KCl-dependent manner. G4 DNA formation was in parallel orientation, which was abolished in the presence of LiCl. The formation of G4 DNA affected both replication and transcription. Finally, immunolocalization of BG4 with MitoTracker confirmed the formation of G-quadruplex in mitochondrial genome. Thus, we characterize the formation of 5 different G-quadruplex structures in human mitochondrial region, which may contribute toward formation of mitochondrial deletions.

DNA G-quadruplex structures: more than simple roadblocks to transcription?

It has been >20 years since the formation of G-quadruplex (G4) secondary structures in gene promoters was first linked to the regulation of gene expression. Since then, the development of small molecules to selectively target G4s and their cellular application have contributed to an improved understanding of how G4s regulate transcription. One model that arose from this work placed these non-canonical DNA structures as repressors of transcription by preventing polymerase processivity. Although a considerable number of studies have recently provided sufficient evidence to reconsider this simplistic model, there is still a misrepresentation of G4s as transcriptional roadblocks. In this review, we will challenge this model depicting G4s as simple ‘off switches’ for gene expression by articulating how their formation has the potential to alter gene expression at many different levels, acting as a key regulatory element perturbing the nature of epigenetic marks and chromatin architecture.

Quantification of ATP in cell by fluorescence spectroscopy based on generalized ratio quantitative analysis model

In this contribution, fluorescence spectroscopy based on G-quadruplex formation was developed for the quantification of ATP in cell. In the absence of ATP, thioflavin T (ThT) dye can combine with the G-rich ATP aptamer to form an ATP aptamer-ThT G-quandruplex complex, resulting in the enhancement of fluorescence intensity; otherwise, fluorescence intensity of the system will weaken with the addition of ATP, because ATP has a strong affinity with G-rich ATP aptamer and can replace ThT to form an ATP aptamer-ATP complex. A calibration model based on generalized ratio quantitative analysis model was employed to mitigate the influence of scatterers and background absorbers in cell suspensions. The proposed fluorescence method was applied to the quantitative analysis of ATP in two type of cell lines, and achieved satisfactory quantitative results with accuracy comparable to that of the reference method–ATP detection kit. The limit of detection and limit of quantification of the proposed method for ATP in cell were estimated to be 0.22 and 0.66 μM, respectively. This proposed fluorescence method is highly simple and rapid, and does not require the use of fluorescent labeling.

Abstract 1246: Anticancer activitiy of indolo[2,3-c]quinoline stabilization of the KRAS promoter G4mid structure

Abstract KRAS is a well validated anti-cancer therapeutic target; many active drug discovery programs are ongoing with foci on individual mutant isoforms, necessitating a wide array of drugs to be developed and missing cancers with dysregulation of non-mutant KRAS. Transcriptional down-regulation has been demonstrated to be lethal to tumor cells with aberrant KRAS signaling, irrespective of mutational status, and to potentially have a wide therapeutic window. G-quadruplex (G4) formation within KRAS's core promoter offers a molecular target to decrease the gene expression. Targeted G4-stabilization within the KRAS promoter targets cancer cells' acquired addiction to the transforming event of aberrant KRAS signaling through an activating mutation or protein overexpression, and thus is applicable to both of these deviant states. Small molecules that specifically inhibit transcriptional expression of KRAS genes have great potential for patients whose tumors have disregulated KRAS function. In the current works, compounds clarify the physiologically relevant G4 within the KRAS promoter (G4mid) through a combination of ex vivo screening, luciferase, cytotoxicity, and transcriptional regulation assays. A small set of novel indolo[2,3-c]quinoline compounds were synthesized and a newly optimized FRET Melt2 assay with a Z' score of >0.5 was used to determine KRAS G4mid stabilization efficacy and selectivity. Compounds were then examined for KRAS promoter stabilization using a luciferase assay, pancreatic and ovarian cancer cytotoxicity by an MTS assay, and correlative changes in KRAS transcription as measured by multiplexed qPCR. JM-222 is the lead indolo[2,3-c]quinoline compound that demonstrates KRAS G4mid selective stabilization and promoter regulation, cancer cell cytotoxicity and altered KRAS transcription. These works highlight the function of G4mid in regulating KRAS transcription, novel synthetic methods for indolo[2,3-c]quinoline synthesis, the improved utility of the FRET Melt2 assay, and the overall potential for small compound-mediated KRAS promoter G4 stabilization for patients harboring tumors with aberrant KRAS signaling. Citation Format: Alexandra Maria Psaras, Jared Miller, L. Nathan Tumey, Tracy A. Brooks. Anticancer activitiy of indolo[2,3-c]quinoline stabilization of the KRAS promoter G4mid structure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1246.

Curcumin arrests G-quadruplex in the nuclear hyper-sensitive III1 element of c-MYC oncogene leading to apoptosis in metastatic breast cancer cells

c-MYC is deregulated in triple negative breast cancer (TNBC) pointing to be a promising biomarker for breast cancer treatment. Precise level of MYC expression is important in the control of cellular growth and proliferation. Designing of c-MYC-targeted antidotes to restore its basal level of cellular expression holds an optimistic approach towards anti-cancer treatment. MYC transcription is dominantly controlled by Nuclear Hypersensitive Element III-1 (NHEIII1) upstream of the promoter region possessing G-Quadruplex silencer element (Pu-27). We have investigated the selective binding-interaction profile of a natural phytophenolic compound Curcumin with native MYC G-quadruplex by conducting an array of biophysical experiments and in silico based Molecular Docking and Molecular Dynamic (MDs) simulation studies. Curcumin possesses immense anti-cancerous properties. We have observed significantly increased stability of MYC-G Quadruplex and thermodynamic spontaneity of Curcumin-MYC GQ binding with negative ΔG value. Transcription of MYC is tightly regulated by a complex mechanism involving promoters, enhancers and multiple transcription factors. We have used Curcumin as a model drug to understand the innate mechanism of controlling deregulated MYC back to its basal expression level. We have checked MYC-expression at transcriptional and translational level and proceeded for Chromatin Immuno-Precipitation assay (ChIP) to study the occupancy level of SP1, Heterogeneous nuclear ribonucleoprotein K (hnRNPK), Nucleoside Diphosphate Kinase 2 (NM23-H2) and Nucleolin at NHEIII1 upon Curcumin treatment of MDA-MB-231 cells. We have concluded that Curcumin binding tends to drive the equilibrium towards stable G-quadruplex formation repressing MYC back to its threshold-level. On retrospection of the synergistic effect of upregulated c-MYC and BCL-2 in cancer, we have also reported a new pathway [MYC-E2F-1-BCL-2-axis] through which Curcumin trigger apoptosis in cancer cells. Communicated by Ramaswamy H. Sarma

Label-free electrochemical homogeneous detection of the depression marker human apolipoprotein A4 based on proximity hybridization triggered rolling circle amplification

In this paper, we developed a label-free homogeneous electrochemical sensor for detection of apolipoprotein A4 based on proximity hybridization triggered rolling circle amplification induced G-quadruplex formation. The presence of apolipoprotein A4 promoted the formation of a proximate complex via the proximity hybridization of the aptamer DNA strands, which unfolded the molecular beacon, the stem part of molecular beacon as a primer to initiate the RCA process. Thus, with the electrochemical indicator hemin selectively intercalated into the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target apolipoprotein A4. Thus, using this “signal-off” mode, label-free homogeneous electrochemical strategy for sensitive apolipoprotein A4 assay with a wide range from 1 pg mL−1 to 100 ng mL−1, with a low detection limit of 0.51 pg mL−1. And it rendered satisfactory analytical performance for the determination of apolipoprotein A4 in serum samples. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. The satisfactory results indicated that the proposed sensor had promising potential in the clinical diagnosis of depression.

Characterization of In Vitro G-Quadruplex Formation of Imetelstat Telomerase Inhibitor

Imetelstat (GRN163L) is a potent and specific telomerase inhibitor currently in clinical development for the treatment of hematological malignancies such as myelofibrosis and myelodysplastic syndrome. It is a 13-mer N3'-P5' thio-phosphoramidate oligonucleotide covalently functionalized at the 5'-end with a palmitoyl lipid moiety through an aminoglycerol linker. As a competitive inhibitor of human telomerase, imetelstat directly binds to the telomerase RNA component sequence (hTR) in the catalytic site of the enzyme and acts as a direct competitor of human telomere binding. Administration of imetelstat causes progressive shortening of the telomeres, thereby inhibiting malignant cells' proliferation. We report here the ability of imetelstat to form stable, parallel, intermolecular G-quadruplex structures in vitro. The impact of the ionic environment on the formation and stability of imetelstat higher-order structure was investigated through circular dichroism spectroscopy, thermal denaturation analysis, and size-exclusion chromatography. We demonstrated that different structural elements, such as the 5'-palmitoyl linker and the thio-phosphoramidate backbone, critically contribute to G-quadruplex stability. Experiments further showed that G-quadruplex formation does not hamper binding to the hTR oligonucleotide sequence in vitro.

Detecting the formation of human c-KIT oncogene promoter G-Quadruplex by Taylor dispersion analysis

The formation of G-quadruplex (G4) structures in oncogenic G-rich promoter regions are implicated in their biological functions, especially the inhibition of transcription. The binding of cations is thought to contribute to the stabilization of the G4 formation and competition against the duplex formation in the genomic sequence. Furthermore, it might affect the recognition of DNA-binding proteins. Therefore, measuring the interaction between G4 DNA and cations in a free solution environment is critical for evaluating G4 DNA biological functions. However, how binding to cations (K+ and NH4+) affects the folding equilibrium of the G4 structure remains unclear. In this work, a Taylor dispersion analysis (TDA) method using a capillary electrophoresis (CE) instrument was established for the quantitative characterization of the cation-dependent G4 formation in the human c-KIT oncogene promoter region, as well as diffusivities and hydrodynamic radii of DNA variations before and after folding. Our results showed that both K+ and NH4+ can induce the random-coiled c-KIT DNA to unfold and form a more unstretched intermediate state and then fold into tightly structured G4s with smaller size. The G4 size induced by NH4+ was smaller than that induced by K+ ions, though these two cations induced the c-KIT G4 DNA formation with similar binding constants (order of magnitude around 106 M−1). The TDA method can be widely used for rapid structural analyses of trace amounts of DNA mixtures, which effectively differentiate DNA variations or DNA-ligand complex conformations.

An increase in DNA G-quadruplex formation in acute myelocytic leukemia is detected by a supramolecular probe

Acute myeloid leukemia (AML) is a common acute leukemia in both adults and children, with poor early detection and diagnosis. Therefore, identifying new indicators for AML detection is significant for effective treatment. Here, we developed a supramolecular probe that exhibits high specificity and sensitivity to G-quadruplex structures in physiological buffer solution, chromosomes, and cells. Using this probe, we tested the DNA extracted from different types of cells and found that the DNA extracted from human acute myeloid leukemia cells HL-60 and KG-1 enhanced the probe fluorescence more significantly than the DNA extracted from other cells. This phenomenon may be related to a large number of G-quadruplexes in acute myeloid leukemia cells, implicating that G-quadruplex levels may be a potential indicator for the detection of acute myeloid leukemia.