Intramolecular Parallel G-quadruplex Research Articles

A sensitive visual DNAzyme-based strategy for Cronobacter sakazakii in PIF by aRPA

Cronobacter sakazakii, as a most important foodborne pathogen in powdered infant formula (PIF), can cause diseases with high mortality in infants and young children, and has become a key monitoring target in dairy industry. In this study, a visual detection strategy based on DNAzyme and asymmetry recombinase polymerase amplification (aRPA) was developed for monitoring C. sakazakii in PIF. A large amount of single-stranded DNA (ssDNA) rich of guanine (G) was produced in this process and ssDNA could bend and fold into intramolecular parallel G-quadruplex in the presence of potassium chloride. The complex of G-quadruplex and hemin, described as DNAzyme, has strong peroxidase-like activity and catalyze the reaction of H2O2 and 3,3′,5,5′-tetramethylbenzidine (TMB), making the color of solution blue. To our knowledge, this is the first attempt in the detection of C. sakazakii based on aRPA and DNAzyme. The limits of detection (LOD) of this method for C. sakazakii were as low as 2.2 CFU mL−1 in pure culture and 5.4 × 101 CFU g−1 in artificially contaminated PIF, respectively. Besides, good stability of the method was shown in the on-site simulation evaluation, which was realized by only warm water, indicating that such strategy may have bright application prospects for field test without heating equipment.

Four-Layered Intramolecular Parallel G-Quadruplex with Non-Nucleotide Loops: An Ultra-Stable Self-Folded DNA Nano-Scaffold.

A four-stranded scaffold of nucleic acids termed G-quadruplex (G4) has found growing applications in nano- and biotechnology. Propeller loops are a hallmark of the most stable intramolecular parallel-stranded G4s. To date, propeller loops have been observed to span only a maximum of three G-tetrad layers. Going beyond that would allow creation of more stable scaffolds useful for building robust nanodevices. Here we investigate the formation of propeller loops spanning more than three layers. We show that native nucleotide sequences are incompatible toward this goal, and we report on synthetic non-nucleotide linkers that form a propeller loop across four layers. With the established linkers, we constructed a four-layered intramolecular parallel-stranded G4, which exhibited ultrahigh thermal stability. Control on loop design would augment the toolbox toward engineering of G4-based nanoscaffolds for diverse applications.

Stabilization of c-KIT G-Quadruplex DNA Structures by the RNA Polymerase I Inhibitors BMH-21 and BA-41.

The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression regulation. We explored the ability of BMH-21 (1) and its analogue BA-41 (2) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3′-end, stabilized by an extensive network of π–π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 106 M−1). Compound BA-41 (2) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound 2 with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.

Characterization of G-Quadruplex Motifs in espB, espK, and cyp51 Genes of Mycobacterium tuberculosis as Potential Drug Targets

G-quadruplex structure forming motifs are among the most studied evolutionarily conserved drug targets that are present throughout the genome of different organisms and susceptible to influencing various biological processes. Here we report highly conserved potential G-quadruplex motifs (PGQs) in three essential genes (espK, espB, and cyp51) among 160 strains of the Mycobacterium tuberculosis genome. Products of these genes are involved in pathways that are responsible for virulence determination of bacteria inside the host cell and its survival by maintaining membrane fluidity. The espK and espB genes are essential players that prevent the formation of mature phagolysosome and antigen presentation by host macrophages. The cyp51 is another PGQ-possessing gene involved in sterol biosynthesis pathway and membrane formation. In the present study, we revealed the formation of stable intramolecular parallel G-quadruplex structures by Mycobacterium PGQs using a combination of techniques (NMR, circular dichroism [CD], and gel electrophoresis). Next, isothermal titration calorimetry (ITC) and CD melting analysis demonstrated that a well-known G-quadruplex ligand, TMPyP4, binds to and stabilizes these PGQ motifs. Finally, polymerase inhibition and qRT-PCR assays highlight the biological relevance of PGQ-possessing genes in this pathogen and demonstrate that G-quadruplexes are potential drug targets for the development of effective anti-tuberculosis therapeutics.

Identification and characterization of G-quadruplex formation within the EP0 promoter of pseudorabies virus

EP0 is an important early gene that modulates the life cycle of pseudorabies virus (PRV). A guanine-rich sequence overlapping with three Sp1 binding sites is located upstream of the transcription start site (TSS) in the EP0 promoter. Using native polyacrylamide gel electrophoresis (PAGE) and circular dichroism (CD), we verified that the G-rich region in the EP0 promoter forms an intramolecular parallel G-quadruplex (G4) in the presence of K+ ions. Further dimethyl sulphate (DMS) footprinting and Taq polymerase stop assays indicates the potential polymorphic folding of G4. In addition, a small chemical ligand, pyridostatin (PDS), promotes and stabilizes the formation of G4. Interestingly, based on the results of electrophoretic mobility shift assays (EMSA), the Sp1 protein bound to G4-bearing DNA with more affinity than DNA lacking the G4 structure. According to the luciferase reporter assay, G4 negatively regulates the EP0 promoter activity. These results demonstrate that Sp1 and G4 cooperate to regulate EP0 promoter activity.

Loop Length Affects Syn-Anti Conformational Rearrangements in Parallel G-Quadruplexes.

A G-quadruplex forming sequence from the MYC promoter region was modified with syn-favoring 8-bromo-2'-deoxyguanosine residues. Depending on the number and position of modifications in the intramolecular parallel G-quadruplex, substitutions with the bromoguanosine analogue at the 5'-tetrad induce conformational rearrangements with concerted all-anti to all-syn transitions for all residues of the modified G-quartet. No unfavorable steric interactions of the C8-substituents in the medium grooves are apparent in the high-resolution structure as determined for a tetrasubstituted MYC quadruplex that exclusively forms the all-syn isomer. In contrast, considerable steric clashes with 5'-phosphate oxygen atoms for those analogues that follow a less flexible 1-nucleotide loop in the native all-anti conformation seem to constitute the major driving force for the tetrad inversion and allow for the rational design of appropriately substituted sequences. Correlations found between the population of species subjected to a tetrad flip and melting temperatures indicate that more effective conformational transitions are compromised by lower thermal stabilities of the modified parallel quadruplexes.

A novel G-quadruplex motif in the Human MET promoter region.

It is known that the guanine-rich strands in proto-oncogene promoters can fold into G-quadruplex structures to regulate gene expression. An intramolecular parallel G-quadruplex has been identified in MET promoter. It acts as a repressor in regulating MET expression. However, the full guanine-rich region in MET promoter forms a hybrid parallel/antiparallel G-quadruplex structure under physiological conditions, which means there are some antiparallel and hybrid parallel/antiparallel G-quadruplex structures in this region. In the present study, our data indicate that g3-5 truncation adopts an intramolecular hybrid parallel/antiparallel G-quadruplex under physiological conditions in vitro. The g3-5 G-quadruplex structure significantly stops polymerization by Klenow fragment in K+ buffer. Furthermore, the results of circular dichroism (CD) spectra and polymerase stop assay directly demonstrate that the G-quadruplex structure in g3-5 fragment can be stabilized by the G-quadruplex ligand TMPyP4 (5,10,15,20-tetra-(N-methyl-4-pyridyl) porphine). But the dual luciferase assay indicates TMPyP4 has no effect on the formation of g3-5 G-quadruplex in HepG2 cells. The findings in the present study will enrich our understanding of the G-quadruplex formation in proto-oncogene promoters and the mechanisms of gene expression regulation.

Effects of the central potassium ions on the G-quadruplex and stabilizer binding

Human telomeres undertake the structure of intra-molecular parallel G-quadruplex in the presence of K+ in eukaryotic cell. Stabilization of the telomere G-quadruplex represents a potential strategy to prevent telomere lengthening by telomerase in cancer therapy. Current work demonstrates that the binding of central K+ with the parallel G-quadruplex is a coordinated water directed step-wise process. The K+ above the top G-tetrad is prone to leak into environment and the 5′-adenine quickly flips over the top G-tetrad, leading to the bottom gate of G-tetrads as the only viable pathway of K+ binding. Present molecular dynamics studies on the two most potent stabilizers RHPS4 and BRACO-19 reveal that the central K+ has little influence on the binding conformations of the bound stabilizers. But without the central K+, either RHPS4 or BRACO-19 cannot stabilize the structure of G-quadruplex. The binding strength of stabilizers evaluated by the MM-PBSA method follows the order of BRACO-19> RHPS4, which agrees with the experimental results. The difference in binding affinities between RHPS4 and BRACO-19 is probably related to the ability to form intramolecular hydrogen bonds and favorable van del Waals interactions with G-quadruplex. In the models that have one central K+ located at the upper/lower binding site, the corresponding top/bottom stacked stabilizers show more favorable binding affinities, indicating the apparent promoting effect of central K+ on the stabilizer binding. Our findings provide further insights into the regulatory effect of K+ on the G-quadruplex targeted binding, which is meaningful to the development of G-quadruplex stabilizers.

An intramolecular G-quadruplex structure formed in the human MET promoter region and its biological relevance.

Previous studies have shown that promoter regions of many proto-oncogenes can fold into G-quadruplexes, which are potentially involved in the regulation of genes. Bioinformatics analysis suggested that there was a G-rich sequence within -48 to -26 region of the human MET promoter (named Pu23WT). In this study, we proved that Pu23WT adopted an intramolecular parallel G-quadruplex structure under physiological conditions in vitro, and the cationic porphyrin TMPyP4 enhanced the stability of the Pu23WT G-quadruplex. To better understand the functions of Pu23WT in the MET expression, we performed a series of analysis on several cancer cells. Experimental data revealed that TMPyP4 treatment attenuated the expression of MET in HepG2, BGC823, and U87MG cells, resulting in the cellular proliferation inhibition, G1 phase cell cycle arrest and cell migration retardation. ChIP assay results indicated that TMPyP4 probably prohibited the Pu23WT G-quadruplex from binding to the activator Sp1, which could be one of the mechanisms that led to the transcription inhibition of MET gene. It is the first study on the G-quadruplex structure in the human MET promoter and its functions in cancer cells. We believe that this structure is a potential target for anticancer treatment.

General peroxidase activity of a parallel G-quadruplex-hemin DNAzyme formed by Pu39WT - a mixed G-quadruplex forming sequence in the Bcl-2 P1 promoter.

BackgroundA 39-base-pair sequence (Pu39WT) located 58 to 19 base pairs upstream of the Bcl-2 P1 promoter has been implicated in the formation of an intramolecular mixed G-quadruplex structure and is believed to play a major role in the regulation of bcl-2 transcription. However, an extensive functional exploration requires further investigation. To further exploit the structure–function relationship of the Pu39WT-hemin DNAzyme, the secondary structure and peroxidase activity of the Pu39WT-hemin complex were investigated.ResultsExperimental results showed that when Pu39WT was incubated with hemin, it formed a uniparallel G-quadruplex-hemin complex in K+ or Na+ solution, rather than a mixed hybrid without bound hemin. Also, Pu39WT-hemin showed peroxidase activity (ABTS2−) in the presence of H2O2 to produce the colored radical anion (ABTS•-), which could then be used to determine the parameters governing the catalytic efficiency and reveal the peroxidase activity of the Pu39WT-hemin DNAzyme.ConclusionsThese results demonstrate the general peroxidase activity of Pu39WT-hemin DNAzyme, which is an intramolecular parallel G-quadruplex structure. This peroxidase activity of hemin complexed with the G-quadruplex-forming sequence in the Bcl-2 gene promoter may imply a potential mechanism of hemin-mediated cellular injury.

Identification, Characterization and Application of a G-Quadruplex Structured DNA Aptamer against Cancer Biomarker Protein Anterior Gradient Homolog 2

BackgroundAnterior gradient homolog 2 (AGR2) is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism.Methodology/Principal FindingsThrough a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with Kd values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB) probe for selective and sensitive detection of AGR2.Conclusion/SignificanceIn this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex structure and its structure and binding affinity are strongly dependent on the nature of the monovalent ion. Furthermore, with our design of AGR2-aMB, AGR2 could be sensitively and selectively detected. This aptamer probe has great potential to serve as a useful tool for early diagnosis and prognosis of cancer and for fundamental research to elucidate the biochemical functions of AGR2.

Formation of an Intramolecular G-Quadruplex of Human Telomere Induced by Poly(l-lysine) under Salt-Deficient Conditions

A single-stranded G-tract human telomere DNA sequence is able to fold into intramolecular G-quadruplex structures which may be important for a number of biological processes and disease-related mechanisms. Poly(L-lysine) (PLL) polymer is linear polypeptides with lysine as the repeat unit and has been employed as a gene carrier in achieving targeted delivery of DNA to cancer cells. To explore the influence of PLL on the conformation of Hum24 DNA, we have investigated the interaction of PLL with Hum24 by biophysical methods, mainly CD, ESI-MS, and polyacrylamide gel electrophoresis for the first time. The CD data have shown that PLL can induce single-stranded Hum24 to form an intramolecular parallel G-quadruplex structure, further confirmed by ESI-MS analysis and gel electrophoresis results. The formation of an intramolecular G-quadruplex is strongly dependent on the Hum24/PLL molar ratios and the length of both the polypeptides and oligonucleotide. Such phenomena may be interpreted by electrostatically attracting negative-charged Hum24 by positive-charged PLL which facilitates the close contact between the guanines and formation of hydrogen bonding, thus leading the final shape of a G-quadruplex structure.

9- N-Substituted berberine derivatives: Stabilization of G-quadruplex DNA and down-regulation of oncogene c- myc

A series of 9- N-substituted berberine derivatives ( 2a– j) were synthesized and evaluated as a new class of G-quadruplex binding ligands. G-quadruplex of DNA had been proven to be the transcription controller of human c-myc gene. The interaction of 9- N-substituted berberine derivatives with G-quadruplex DNA in c-myc was examined via EMSA, CD spectroscopy, FRET-melting method, PCR-stop assay, competitive dialysis, cell proliferation assay, and RT-PCR assay. The experiment results indicated that these derivatives could selectively induce and stabilize the formation of intramolecular parallel G-quadruplex in c-myc, which led to down-regulation of transcription of the c-myc in the HL60 lymphomas cell line. The related structure–activity relationships were also discussed.

G-quadruplex formation within the promoter of the KRAS proto-oncogene and its effect on transcription

In human and mouse, the promoter of the KRAS gene contains a nuclease hypersensitive polypurine–polypyrimidine element (NHPPE) that is essential for transcription. An interesting feature of the polypurine G-rich strand of NHPPE is its ability to assume an unusual DNA structure that, according to circular dichroism (CD) and DMS footprinting experiments, is attributed to an intramolecular parallel G-quadruplex, consisting of three G-tetrads and three loops. The human and mouse KRAS NHPPE G-rich strands display melting temperature of 64 and 73°C, respectively, as well as a K+-dependent capacity to arrest DNA polymerase. Photocleavage and CD experiments showed that the cationic porphyrin TMPyP4 stacks to the external G-tetrads of the KRAS quadruplexes, increasing the Tm by ∼20°C. These findings raise the intriguing question that the G-quadruplex formed within the NHPPE of KRAS may be involved in the regulation of transcription. Indeed, transfection experiments showed that the activity of the mouse KRAS promoter is reduced to 20% of control, in the presence of the quadruplex-stabilizing TMPyP4. In addition, we found that G-rich oligonucleotides mimicking the KRAS quadruplex, but not the corresponding 4-base mutant sequences or oligonucleotides forming quadruplexes with different structures, competed with the NHPPE duplex for binding to nuclear proteins. When vector pKRS-413, containing CAT driven by the mouse KRAS promoter, and KRAS quadruplex oligonucleotides were co-transfected in 293 cells, the expression of CAT was found to be downregulated to 40% of the control. On the basis of these data, we propose that the NHPPE of KRAS exists in equilibrium between a double-stranded form favouring transcription and a folded quadruplex form, which instead inhibits transcription. Such a mechanism, which is probably adopted by other growth-related genes, provides useful hints for the rational design of anticancer drugs against the KRAS oncogene.

Design and Synthesis of an Expanded Porphyrin That Has Selectivity for the c-MYC G-Quadruplex Structure

Cationic porphyrins are known to bind to and stabilize different types of G-quadruplexes. Recent studies have shown the biological relevance of the intramolecular parallel G-quadruplex as a transcriptional silencer in the c-MYC promoter. TMPyP4 also binds to this G-quadruplex and most likely converts it to a mixed parallel/antiparallel G-quadruplex with two external lateral loops and one internal propeller loop, suppressing c-MYC transcriptional activation. To achieve therapeutic selectivity by targeting G-quadruplexes, it is necessary to synthesize drugs that can differentiate among the different types of G-quadruplexes. We have designed and synthesized a core-modified expanded porphyrin analogue, 5,10,15,20-[tetra(N-methyl-3-pyridyl)]-26,28-diselenasapphyrin chloride (Se2SAP). Se2SAP converts the parallel c-MYC G-quadruplex into a mixed parallel/antiparallel G-quadruplex with one external lateral loop and two internal propeller loops, resulting in strong and selective binding to this G-quadruplex. A Taq polymerase stop assay was used to evaluate the binding of TMPyP4 and Se2SAP to G-quadruplex DNA. Compared to TMPyP4, Se2SAP shows a greater selectivity for and a 40-fold increase in stabilization of the single lateral-loop hybrid. Surface plasmon resonance and competition experiments with duplex DNA and other G-quadruplexes further confirmed the selectivity of Se2SAP for the c-MYC G-quadruplex. Significantly, Se2SAP was found to be less photoactive and noncytotoxic in comparison to TMPyP4. From this study, we have identified an expanded porphyrin that selectively binds with the c-MYC G-quadruplex in the presence of duplex DNA and other G-quadruplexes.