5'-tiRNA-Gln inhibits hepatocellular carcinoma progression by repressing translation through the interaction with eukaryotic initiation factor 4A-I / 医学前沿

tRNA-derived small RNAs (tsRNAs) are novel non-coding RNAs that are involved in the occurrence and progression of diverse diseases. However, their exact presence and function in hepatocellular carcinoma (HCC) remain unclear. Here, differentially expressed tsRNAs in HCC were profiled. A novel tsRNA, tRNAGln-TTG derived 5'-tiRNA-Gln, is significantly downregulated, and its expression level is correlated with progression in patients. In HCC cells, 5'-tiRNA-Gln overexpression impaired the proliferation, migration, and invasion in vitro and in vivo, while 5'-tiRNA-Gln knockdown yielded opposite results. 5'-tiRNA-Gln exerted its function by binding eukaryotic initiation factor 4A-I (EIF4A1), which unwinds complex RNA secondary structures during translation initiation, causing the partial inhibition of translation. The suppressed downregulated proteins include ARAF, MEK1/2 and STAT3, causing the impaired signaling pathway related to HCC progression. Furthermore, based on the construction of a mutant 5'-tiRNA-Gln, the sequence of forming intramolecular G-quadruplex structure is crucial for 5'-tiRNA-Gln to strongly bind EIF4A1 and repress translation. Clinically, 5'-tiRNA-Gln expression level is negatively correlated with ARAF, MEK1/2, and STAT3 in HCC tissues. Collectively, these findings reveal that 5'-tiRJNA-Gln interacts with EIF4A1 to reduce related mRNA binding through the intramolecular G-quadruplex structure, and this process partially inhibits translation and HCC progression.

Analysis of the Factors Affecting the Configuration of the Anti-parallel G-quadruplex Formed by 22-nt Telomere RNA Fragments / 中国生物化学与分子生物学报

G-quadruplexes with different conformations often exhibit different binding abilities to proteins and play different physiological functions. It is of great significance to analyze the factors affecting the configuration of G-quadruplex to explore its physiological function. Different from DNA, RNA sequences have shown parallel G-quadruplex structures in previous reports. However, we found that the only RNA sequence, the 22-nt telomere RNA fragment (ORN-N, A

Effect of Palmatine Binding to MYC G-quadruplex on Proliferation and Apoptosis of Colon Cancer Cells and Molecular Mechanism / 中国实验方剂学杂志

Objective:To explore whether palmatine interferes with the proliferation and apoptosis of colon cancer HCT116 cells by binding to G-quadruplex in the promoter region of MYC proto-oncogene and its possible molecular mechanism. Method:Fluorescence spectrum was used to analyze the binding ability of palmatine to MYC G-quadruplex. Circular dichroism analysis was conducted to confirm the effect of palmatine on the configuration of MYC G-quadruplex， followed by the prediction of their binding mode based on molecular docking and the localization analysis of palmatine in HCT116 cells under a fluorescence microscope. The effects of palmatine on MYC gene transcription and MYC protein expression were determined by real-time fluorescence quantitative polymerase chain reaction and Western blot， respectively. The effects of palmatine on the viability and apoptosis of HCT116 cells were further assayed by thiazolyl blue tetrazolium bromide （MTT） assay and flow cytometry. Result:As revealed by fluorescence spectrum and molecular docking analysis， palmatine might bind to G-quadruplex of MYC gene through stacking. Circular dichroism analysis showed that palmatine could maintain the parallel configuration of MYC<italic> </italic>G-quadruplex. It was discovered in fluorescence imaging that palmatine was distributed in the nucleus and bond to G-quadruplex of MYC gene. In addition， palmatine inhibited MYC gene transcription， MYC protein expression， as well as the viability of HCT116 cells， and promoted the apoptosis of HCT116 cells. Conclusion:Palmatine is able to bind to MYC G-quadruplex to further inhibit the expression of MYC gene and protein expression， which may be one of the molecular mechanisms of palmatine in suppressing the proliferation of colon cancer HCT116 cells and facilitating their apoptosis.

G-quadruplex Structure in miRNA / 中国生物化学与分子生物学报

G-quadruplex (G4) is a non-canonical nucleic acid secondary structure composed of guanine-rich DNA or RNA sequences. In the past few decades, researchers have focused on the G-rich sequences in gene promoter region, UTR, telomere and other common gene functional regions, to understand the relationship between their structure and function. In recent years, due to the in-depth study of non-coding RNA in diseases, G-rich sequences in non-coding RNA, especially miRNA, have attracted more attention. G-rich sequence has been involved in all physiological processes of miRNA synthesis, from primary miRNA (pri-miRNA), precursor miRNA (pre-miRNA) to mature miRNA: the dynamic balance formed between the G-quadruplex and the RNA stem-loop structure affects the maturation process of primary and precursor miRNA, causing the change of mature miRNA and the complementary pairing between miRNA and mRNA. This review focused on the dynamic changes between G4 and hairpin structure, and expounded the role of G-rich sequence in pri-miRNA, pre-miRNA, mature miRNA and mRNA. The important role of G-quadruplex in the maturation and function of miRNA was also discussed. It was concluded that the changes of external regulatory factors such as ionic conditions (K+, Mg +, K+ + Mg, Li +Mg +, etc) and G-quadruplex ligand molecules (destabilizer TMPyP4, etc) could regulate the balance between G-quadruplex and stem-loop structure, thus modulating the functions of miRNA. This review may help providing ideas and directions for the study of G-quadruplex function and regulation.

Effect of parthenolide on proliferation and migration of MCF-7 breast cancer cells by targeting the c-myc G-quadruplex / 药学学报

This research investigated the effect of parthenolide on the proliferation and migration of human breast cancer cells and explored the molecular mechanism of that effect. Surface plasmon resonance and fluorescence resonance energy transfer melting were used to detect the binding and stabilizing ability of PTL and G-quadruplex. MTT assays were used to determine the effect of PTL on the proliferation of MCF-7 breast cancer cells. A wound healing assay was performed to detect the migration of MCF-7. The results indicate that PTL shows good binding and stabilizing activities with c-myc G-quadruplex with a KD = 13.1 μmol·L-1. PTL inhibited the proliferation of MCF-7 cells with an IC50 of 21.3 μmol·L-1 (24 h), 14.5 μmol·L-1 (48 h) and 9.1 μmol·L-1 (72 h). PTL inhibited MCF-7 breast cancer cell proliferation and migration and down-regulated the transcription and expression level of c-myc by targeting G-quadruplex.

Screening of G-quadruplex ligands from Macleaya cordata extract by contrast ultrafiltration with liquid chromatography-mass spectrometry and molecular docking / 中国中药杂志

G-quadruplex DNA has become an important target for tumor therapy and anti-tumor development. Modern pharmacology has proved that Macleaya cordata has anti-inflammatory, antibacterial, anti-tumor and other pharmacological effects. Affinity ultrafiltration method can screen active ingredients from compounds rapidly, but G-quadruplex DNA ligands are difficult to dissociate, which is a key step in conventional ultrafiltration method. In this paper, the filtrates after ultrafiltration were determined by HPLC-MS in substitution. The peaks with 20% reduction of MS response from the incubation vs control were considered to be ligand components to G-quadruplex. Two of the peaks with the relative abundance above 30% were identified as sanguinarine(SAN) and chelerine(CHE). Their circular dichroism conformations further proved that SAN and CHE are active ligands of HT4. In addition, another two gradients with high relative abundance were identified as protopine(PRO) and allpcryprotopine(ALL). The binding rate of SAN, CHE, PRO and ALL was calculated according to the HPLC-MS results, and the results showed a consistency with that of the molecular docking method. The proposed method can be used to screen active components from mixture.

Application of atomic force microscopy-based single molecule force spectroscopy in G-quadruplex studies / 南方医科大学学报

Telomere plays a crucial role in the physiological and pathological processes of cells. At the end of the telomere, the single-stranded DNA repeat sequence rich in guanine (G) folds in the presence of monovalent metal ions such as Na or K to form a G-quadruplex structure. This structure can not be extended by telomerase and inhibits the activity of telomerase, thus becoming a potential anticancer target. Stabilizing the formation of DNA G-quadruplex structures by small molecule ligands has become a new strategy for designing many anticancer drugs, and studying the interaction strength of these small molecule ligands with G-quadruplex is thus of particular importance for screening highly effective anticancer drugs. Single molecule force spectroscopy enables direct measurement of the interaction between small molecule ligands and G-quadruplexes. This review highlights the advances of single-molecule force spectroscopy based on atomic force microscopy in the study of the G quadruplex structure and its interaction with small molecule ligands, and summarizes the application and development trend of single molecule force spectrum technology in G quadruplex.

Establishment and Application of Functional Nucleic Acid Biosensor for Detection of Mercury Ion / 分析化学

Excessive mercury ions have negative effects on individual health. So, it is of great significance to develop a method for rapid, sensitive and specific detection of Hg2+. In this study, the method for detection of Hg2+ was developed based on specific T-Hg-T mismatche and G-quadruplex. The template sequence mainly included the recognition region which could combine with Hg2+ specifically, the rich G region which could form G-quadruplex, and the speacer 18 partition zone. The target sequence mainly included 5' ends combining area and 3' end T-rich region. Templates and targets could be combined and the elongation was triggered only in the presence of Hg2+. Furthermore, the G-rich sequences were stripped off the templates and could form a single-stranded structure, because Spacer 18 had partition function. The G-quadruplex was formed with the K+and hemin, and catalyzed H2 O2-2,2-azinobis (3-ethylbenzothiozoline)-6-sulfonic acid(ABTS) reaction with color variations. The detection could be done within 35 min, and the Hg2+ concentration exhibited a linear correlation with absorbance at 414 nm within range of 20-500 nmol/ L, with a detection limit of 16. 5 nmol/ L (3σ). The recoveries of Hg2+ spiked in tap water were 98. 5％ - 103. 5％ . The method exhibited the advantages such as simple operation, low cost and short time, and operation value in emergency treatment and real-time environmental detection.

Pb2+-Induced Allosteric PS2.M as A Label-Free Colorimetric Sensor for Detection of Cumulative Radon Radiation / 分析化学

A new sensitive method for detection of cumulative radon radiation based on the lead was established. PS2.M which stabilized by K+with hemin as a co-factor exhibits superior peroxidase-like activity, and can effectively catalyze the H2O2-mediated oxidation of TMB. In the presence of Pb2+, K+-stabilized PS2.M DNAzymes are induced to undergo a conformational change,because Pb2+has a higher efficiency with regard to stabilizing G-quadruplexes than K+, accompanied by a decrease of catalytic activity and a sharp decrease of readout signal. In the work, a novel"turn-off"model of colorimetry-Pb2+biosensor based on superior peroxidase-like activity of G-quadruplex for Pb2+analysis was developed. The fading degree of reaction system (△A value) was linearly related to Pb2+concentration in the range of 5. 0×10-9-1. 8× 10-7mol/L. The linear regression equation was △A=0.36+0.13C (10-8mol/L), with R=0.9987. The detection limits of lead and radon were 3.76 nmol/L(S/N=3) and 1.96×103Bq·h/m3(S/N=3), respectively. The method exhibited good selectivity, high sensitivity and convenient operation, and could avoid the radioactive hazard in determination of the radon in environment.

A Fluorescence Biosensor for Detection of Mercury Ion Based on Oligonucleotide / 分析化学

A simple,fast and highly sensitive fluorescence analysis method for detection of mercury ion was developed based on N-methyl-mesoporphyrin IX (NMM)/G-quadruplex DNA system and specific T-Hg-T mismatches.In this strategy,a large number of thymine was introduced into guanine-rich oigonucleotides which could form G-quadruplex.In the presence of Hg2+,guanine-rich oigonucleotides and complementary strand could form double-stranded DNA molecule by specific T-Hg-T mismatch pair,leading to destruction of G-quadruplex DNA structure.In the absence of Hg2+,guanine-rich oigonucleotides spontaneously formed G-quadruplex DNA structure that could bound NMM to generate intense fluorescence.Based on the above facts,a sensitive fluorescence biosensor for determination of Hg2+ was fabricated.And the optimal conditions for Hg2+ determination were as follows:buffer solution pH of 6.7,20 mmol/L KCl and 2.5 μmol/L NMM in buffer and incubation for 2 h.Under the optimal conditions,the fluorescence intensity signal change (F0-F) and the Hg2+ concentration exhibited a linear correlation within 50 nmol/L to 1000 nmol/L range with a low detection limit of 22.8 nmol/L (3σ).The biosensor exhibited good selectivity toward common metal ions.The developed method was successfully employed to detect Hg2+ in tap water with recovery of 106.1％-107.8％.

The G4-quadruplex mutation of the core gene enhances the immunogenicity of hepatitis C virus DNA vaccine / 中国生化药物杂志

Objective To investigate the effect of G-quadruplex (G4) RNA structure of core of hepatitis C virus (HCV) on the specific immune response. Methods Circular dichroism (CD) was usedto detect the G4 spatial structure of the G4 oligonucleotide chain RNA (named as G4R) and its mutant of G4 (named as G4RM) by G base site-specific mutation.The HCV wild-type core gene G4(DNA) sequence was mutated as G4M-core by PCR site-directed mutagenesis without changing the amino acid codon.Then wild type and mutated core genes were constructed into the eukaryotic expression plasmid pcDNA3.1-Myc, and produced as pcDNA3.1-core-G4-WT (named as pG4) and pcDNA3.1-core-G4-M (named as pG4M), the expression of core protein was examined by Western blot. The mice were immunized with the pG4 and pG4M plasmids DNA respectively, and their humoral and cellular responses were examined. Results CD results showed that the structure of G4RM was changed compared to Wild type G4R, and the melting curve analysis showed the melting temperature of GR4M was lower than that of G4R, which indicates that G4RM structure is unstable. Western blot analysis showed that pG4M had much higher protein expression level compared to pG4(P<0.05). Analysis of animal immunization showed that pG4M induced increased levels of total IgG and IFN-γ compared to pG4(P<0.05). The IgG level of the pG4M group was 1.61 times higher than that of the pG4 group. By enzyme-linked immunospot(ELISpot)assay, we found that the release IFN-γ level of pG4M was 1.39 times higher than those of pG4. Flow cytometry showed that the intracellular IFN-γ production in the splenic CD4+ T cells was 1.79 times than those of pG4. Conclusion The G-quadruplex structure of HCV core can inhibit its protein translation. The mutation of G-quadruplex of core led to increased Th1-type immune responses. This is the first report demonstrate that HCV core G-quadruplex mutation can enhance its immunogenicity and could be used as a new strategy ofexploring HCV vaccine with enhanced immunogenicity.

Design, synthesis and biological activities of novel 3,6-diamide xanthones / 药学学报

A series of novel xanthones with terminal amine substituents at xanthone's C3 and C6 positions were designed and synthesized as potential ligands for telomeric G-quadruplex DNA. All the compounds in this series were bound to telomeric G-quadruplex in a "thread intercalation" manner that illustrated both in molecular docking and spectrometric studies. Among them, 10c and 10d showed better binding abilities and specific affinity toward G-quadruplex DNA HTG21 over ctDNA in the fluorescence assay. The antiproliferative activities of four screened compounds were examined in three cancer cells by MTT in vitro, and their inhibitory effects were observed at low micromolar ranges. In addition, the PCR stop assay demonstrated that 10c and 10d effectively inhibited the amplification ability of telomerase.

G-quadruplex Structures in Promoters of MET Proto-oncogene / 中国医科大学学报

Objective To identify G?quadruplex structures in the promoter region of MET. Methods CD spectroscopy,UV spectroscopy,non?denatured electrophoresis and PCR stop assay were applied to indicate the G?quadruplex structure and its function. Results The Pu23WT se?quence in the promoter of MET adopted an intramolecular parallel G?quadruplex structure under physiological conditions in vitro,which can stop the extension of Pmet. Conclusion G?quadruplex structure in the promoter might inhibit MET gene expression in vivo.

Study of human telomere G4-DNA stabilizers / 中国药理学通报

Telomere maintenance plays a critical role in cancer progression.Approximately 85% human cancer cells maintain their telomere length through activation of telomerase.Other 15%of cancers maintain telomere length independently of telom-erase by alternative lengthening of telomeres (ALT)pathway. Both events are equally important for telomere length mainte-nance of cancer cells.Human telomere consists of a series of G rich DNA sequences,which could form G-quadruplex.The for-mation of this structure can block the extension of telomeres by telomerase or ALT,resulting in cancer cell death.Thereby,G-quadruplex has been one of the focuses of anticancer therapy in recent years.This review focuses on the latest progress of G-quadruplex stabilizers.

Electroluminescence Biosensor for Determination of Pb2+Based on G-Quadruplex of Aptamer probe / 分析化学

A turn on model of electrochemiluminescence ( ECL)-Pb2+ biosensor based on G-quadruplex of aptamer probe was developed by using quantum dots ( QDs) as labels. The ECL-Pb2+ biosensor was prepared by immobilizing a hairpin aptamer probe with a thiol group at 5 '-end and an amino group at 3 '-end on gold electrode via an Au-S bond. In the presence of Pb2+, the G-rich hairpin aptamer probe opened the stem-loop and formed G-quadruplex, which exposed the amino group at 3'-end and then covalently linked to the carboxyl group on the surface of CdS quantum dots. The fabrication and reaction process of the Pb2+ biosensor were characterized. The responsive ECL intensity was linearly related to the Pb2+ concentration in the range of 2. 0×10-10-5. 0×10-8 mol/L, with the detection limit of 3. 74×10-11 mol/L. In addition, the ECL biosensor exhibited high sensitivity, good selectivity, satisfying stability, and repeatability.

Detection of Lead Ions in Water Based on Surface Energy Transfer between Gold Nanoparticles and Fluorescenct Dyes / 分析化学

We established a sensitive method for the detection of lead ions in aqueous solution based on the surface energy transfer between gold nanoparticles and fluorescent dyes. The fluorescein-modified thrombin aptamer (5′-FAM-GGT TGG TGT GGT TGG-3′) can be selectively transformed to G-quadruplex induced by lead ions. The conformation changes of the aptamer can alter the distance between the energy donor of fluorescent dyes and the energy receptor of gold nanoparticles, and further enhance the fluorescence intensity. The fluorescence recovery efficiency ( F/F0 ) is proportional to the concentration of lead ions in the range of 12. 5-100 nmol/L following the linear regression equation of y=0. 910﹢0. 007c(R2=0. 997)and the limit of determination is about 10 nmol/L. The proposed method has been applied to the determination of lead ions in tap water with satisfied results.

Sensor Design Based on Structure Adjustment in Loops of G-quadruplex / 分析化学

The effects of linking loop structure between guanine ( G) repeats on G-quadruplex formation were investigated. The results show that the unfavorable effects of long linking loops on G-quadruplex formation can be overcome by introducing double-stranded structures in linking loop regions. This finding provides a new way for sensor design. The activity of G-quadruplex DNAzyme can be tuned by utilizing target-mediated formation of double-stranded structures in loops. As an example, T-T mismatches are introduced in loops to destroy double-stranded structures. The stabilization of Hg2+ to T-T mismatches promotes the reformation of double-stranded structures. Correspondingly, the oligonucleotide folds into G-quadruplex, which binds with hemin to form peroxidase-like G-quadruplex DNAzyme. Hg2+ sensor is designed based on this principle. Using this method, Hg2+ quantitation is achieved in the concentration range of 10-700 nmol/L, with a detection limit of 8. 7 nmol/L. Cysteine will compete with T bases to bind with Hg2+, releasing Hg2+from T-Hg2+-T base pairs. Thus cysteine can also be quantified with this system in the concentration range of 20-700 nmol/L, with a detection limit of 14 nmol/L.

DNA, RNA G-quadruplcx and their small molecular ligands: Research advances / 国际药学研究杂志

In recent years, G-quadruplexes overexpression has been found in different biologically significant regions, such as oncogene-promoter regions, human telomeres DNA and mRNA 5'-untranslated region(lJTR). Therefore, a novel anticancer strategy may be developed by searching some ligands to stabilize or to induce the G-quadruplexs so as to inhibit cancer gene transcription and traaslation process. The current structural database of folding topologies. The anticancer mechanism taigeting different types of G-quadruplex, and the progresses on the corresponding small molecular ligands as potent antitumor agents are summarized in this review. © 2006 Editorial office of Foreign Medical Sciences.

Antitumor effect of Macleaya cordata and its molecular mechanism on inducement of human telomeric DNA to form G-quadruplex / 中草药

Objective: To study the antitumor molecular mechanism started on sanguinarine (San) and chelerythrine (Che) from Macleaya cordata. Methods: Determining the IC50 values of San and Che against A-549, HCT-8, and Bel-7402 cell lines by using MTT to clarify whether these two alkaloids are the active components in M. cordata; studying the interaction between human telomeric DNA and two alkaloids respectively by using UV-Vis, FL, and CD methods. Results: San and Che are the active antitumor components of M. cordata; San could induce HT4 to form antiparallel G-quadruplex completely with Ka of 5×108 and Che could induce HT4 to form antiparallel G-quadruplex partially with Ka of 930. Conclusion: One of the antitumor molecular mechanisms of M. cordata is that the two active components could induce human telomeric DNA to form G-quadruplex.

Progress in the study of G-quadruplex telomerase inhibitors / 中国药理学通报

Telomerase can inhibit the shortening of telomere and stimulate continuous cell proliferation to form tumor.The activity of telomerase can be prohibited by G-quadruplex formed by the single chain in G-rich field of telomere 3' end, resulting in tumor cell apoptosis.Ligands which can induce the formation or stabilize the structure of G-quadruplex in G-rich field of oncogene exhibit antitumor function. Nowadays it becomes the core concern of chemists and biologists to screen and structurally design the compounds targeting G-quadruplex.This paper summarized the discoveries in the G-quadruplex-targeted telomerase inhibitors in recent years.