Ligand Binding to Dynamically Populated G-Quadruplex DNA

Several small-molecule ligands specifically bind and stabilize G-quadruplex (G4) nucleic acid structures, which are considered to be promising therapeutic targets. G4s are polymorphic structures of varying stability, and their formation is dynamic. Here, we investigate the mechanisms of ligand binding to dynamically populated human telomere G4 DNA by using the bisquinolinium based ligand Phen-DC3 and a combination of single-molecule FRET microscopy, ensemble FRET and CD spectroscopies. Different cations are used to tune G4 polymorphism and folding dynamics. We find that ligand binding occurs to pre-folded G4 structures and that Phen-DC3 also induces G4 formation in unfolded single strands. Following ligand binding to dynamically populated G4s, the DNA undergoes pronounced conformational redistributions that do not involve direct ligand-induced G4 conformational interconversion. On the contrary, the redistribution is driven by ligand-induced G4 folding and trapping of dynamically populated short-lived conformation states. Thus, ligand-induced stabilization does not necessarily require the initial presence of stably folded G4s.     

Hidden Information Revealed Using the Orthogonal System of Nucleic Acids

In this study, the organization of genetic information in nucleic acids is defined using a novel orthogonal representation. Clearly defined base pairing in DNA allows the linear base chain and sequence to be mathematically transformed into an orthogonal representation where the G–C and A–T pairs are displayed in different planes that are perpendicular to each other. This form of base allocation enables the evaluation of any nucleic acid and predicts the likelihood of a particular region to form non-canonical motifs. The G4Hunter algorithm is currently a popular method of identifying G-quadruplex forming sequences in nucleic acids, and offers promising scores despite its lack of a substantial rational basis. The orthogonal representation described here is an effort to address this incongruity. In addition, the orthogonal display facilitates the search for other sequences that are capable of adopting non-canonical motifs, such as direct and palindromic repeats. The technique can also be used for various RNAs, including any aptamers. This powerful tool based on an orthogonal system offers considerable potential for a wide range of applications.     

Design and Synthesis of Xanthone Analogues Conjugated with Aza-aromatic Substituents as Promising G-Quadruplex Stabilizing Ligands and their Selective Cancer Cell Cytotoxic Action

We have examined the stabilization of higher-order noncanonical G-quadruplex (G4) DNA structures formed by the G-rich sequences in the promoter region of oncogenes such as c-MYC, c-KIT, VEGF and BCl2 by newly synthesized, novel nitrogen-containing aromatics conjugated to xanthone moiety. Compounds with N-heterocyclic substituents such as pyridine (XNiso), benzimidazole (XBIm), quinoxaline (XQX) and fluorophore dansyl (XDan) showed greater effectiveness in stabilizing the G4 DNA as well as selective cytotoxicity for cancer cells (mainly A549) over normal cells both in terms of UV-Vis spectral titrations and cytotoxicity assay. Both fluorescence spectral titrimetric measurements and circular dichroism (CD) melting experiments further substantiated the G4 stabilization phenomenon by these small-molecular ligands. In addition, these compounds could induce the formation of parallel G4 structures in the absence of any added salt condition in Tris ⋅ HCl buffer at 25 °C. In a polymerase stop assay, the formation of stable G4 structures in the promoter of oncogenes and halting of DNA synthesis in the presence of the above-mentioned compounds was demonstrated by using oncogene promoter as the DNA synthesis template. Apoptosis-mediated cell death of the cancer cells was proved by Annexin V-PI dual staining assay and cell-cycle arrest occurred in the S phase of the cell cycles. The plausible mode of binding involves the stacking of the xanthone core on the G4 DNA plane with the possibility of interaction with the 5’-overhang as indicated by molecular dynamics simulation studies.     

Applications of Isothermal Titration Calorimetry in Biophysical Studies of G-quadruplexes

G-quadruplexes are higher-order nucleic acids structures formed by G-rich sequences that are stabilized by tetrads of hydrogen-bonded guanine bases. Recently, there has been growing interest in the study of G-quadruplexes because of their possible involvement in many biological processes. Isothermal titration calorimetry (ITC) has been proven to be a useful tool to study the energetic aspects of G-quadruplex interactions. Particularly, ITC has been applied many times to determine the thermodynamic properties of drug-quadruplex interactions to screening among various drugs and to address drug design. In the present review, we will focus on the ITC studies of G-quadruplex structures and their interaction with proteins and drugs and the most significant results will be discussed.     

CNBP Binds and Unfolds In Vitro G-Quadruplexes Formed in the SARS-CoV-2 Positive and Negative Genome Strands

The Coronavirus Disease 2019 (COVID-19) pandemic has become a global health emergency with no effective medical treatment and with incipient vaccines. It is caused by a new positive-sense RNA virus called severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). G-quadruplexes (G4s) are nucleic acid secondary structures involved in the control of a variety of biological processes including viral replication. Using several G4 prediction tools, we identified highly putative G4 sequences (PQSs) within the positive-sense (+gRNA) and negative-sense (−gRNA) RNA strands of SARS-CoV-2 conserved in related betacoronaviruses. By using multiple biophysical techniques, we confirmed the formation of two G4s in the +gRNA and provide the first evidence of G4 formation by two PQSs in the −gRNA of SARS-CoV-2. Finally, biophysical and molecular approaches were used to demonstrate for the first time that CNBP, the main human cellular protein bound to SARS-CoV-2 RNA genome, binds and promotes the unfolding of G4s formed by both strands of SARS-CoV-2 RNA genome. Our results suggest that G4s found in SARS-CoV-2 RNA genome and its negative-sense replicative intermediates, as well as the cellular proteins that interact with them, are relevant factors for viral genes expression and replication cycle, and may constitute interesting targets for antiviral drugs development.     

Development of Minimal Diguanosinyl Motif toward RNA G-Quadruplex-Like Structures in Solution

Among the non-canonical structures of B-DNA, the G-quadruplex is of particular interest because of its well-defined conformation, high stability, and versatility. Herein we report our studies on the development of an amide-linked minimal diguanosinyl motif that forms a G-quadruplex-like structure in solution in the presence of potassium cations; various linear guanosine amino acid dimers were synthesized with linkers of different chain lengths to investigate the optimum flexibility required to form such structures.     

鸟嘌呤四链体DNAzyme在微生物、生物小分子和核酸检测中的研究进展

鸟嘌呤四链体脱氧核酶(deoxyribozyme,DNAzyme)是一种具有类过氧化物酶催化活性的脱氧核酶,广泛应用于微生物和核酸的分析与检测中。鸟嘌呤四链体DNAzyme在H_2O_2存在时可以催化氧化ABTS、TMB等底物,结合PCR、等温扩增技术以及生物传感器等多种核酸靶标信号放大技术实现待测靶标的便捷灵敏的可视化检测,因此受到了国内外研究者们的高度关注。优化鸟嘌呤四链体的序列和环境因素,能够提高该DNAzyme的催化活性,有助于提高可视化检测方法的灵敏度。本文主要对鸟嘌呤四链体DNAzyme的结构与酶学性质及其应用于微生物、生物毒素和疾病标志物等的基于核酸的检测策略进行综述,为进一步推动鸟嘌呤四链体DNAzyme的广泛应用提供理论基础和技术支持。     

Detection of CpG Methylation in G-Quadruplex Forming Sequences Using G-Quadruplex Ligands

Genomic DNA methylation is involved in many diseases and is expected to be a specific biomarker for even the pre-symptomatic diagnosis of many diseases. Thus, a rapid and inexpensive detection method is required for disease diagnosis. We have previously reported that cytosine methylation in G-quadruplex (G4)-forming oligonucleotides develops different G4 topologies. In this study, we developed a method for detecting CpG methylation in G4-forming oligonucleotides based on the structural differences between methylated and unmethylated G4 DNAs. The differences in G4 topologies due to CpG methylation can be discriminated by G4 ligands. We performed a binding assay between methylated or unmethylated G4 DNAs and G4 ligands. The binding abilities of fluorescent G4 ligands to BCL-2, HRAS1, HRAS2, VEGF G4-forming sequences were examined by fluorescence-based microtiter plate assay. The differences in fluorescence intensities between methylated and unmethylated G4 DNAs were statistically significant. In addition to fluorescence detection, the binding of G4 ligand to DNA was detected by chemiluminescence. A significant difference was also detected in chemiluminescence intensity between methylated and unmethylated DNA. This is the first study on the detection of CpG methylation in G4 structures, focusing on structural changes using G4 ligands.     

电喷雾质谱法研究Kras基因启动子区G-四链体的形成与性质

采用电喷雾质谱（ESI-MS）和圆二色谱（CD）法研究了Kras基因G-四链体的形成与性质,结果表明,在阳离子条件下,KS1序列形成的是平行构象为主的混合构象G-四链体。实验探索了不同位置G碱基突变对Kras原生型序列G-四链体结构的影响,发现在KS1序列中,第6位G碱基突变对G-四链体影响不大;第13位G碱基突变会导致G-四链体层数减少;而第18、19位G碱基突变会导致G-四链体稳定性下降,同时G-四链体构象也向反平行移动,说明这些位置的G碱基对KS1序列G-四链体的形成有较大的贡献,它们很有可能参与了G-四分体平面的组成,最终形成了特殊的G-四链体结构。虽然不同位置的碱基突变对KS1序列G-四链体的影响各不相同,但突变后的序列形成G-四链体的能力整体比原生型序列弱,这在一定程度上表明,生物体内一部分基因突变后所导致的癌症可能与G-四链体结构的破坏有关。该结果可为G-四链体的结构和生物学功能研究提供更多信息,同时也可为相关抗癌药物的研制提供更广阔的思路和方向。