G-quadruplexes (G4s) within the human genome have undergone extensive molecular investigation, with a strong focus on telomeres, gene promoters and repetitive regulatory sequences. G4s play central roles in regulating essential biological processes, including telomere maintenance, replication, transcription and translation. Targeting these molecular processes with G4-binding ligands holds substantial therapeutic potential in anticancer treatments and has also shown promise in treating neurological, skeletal and muscular disorders. The presence of G4s in bacterial and viral genomes also suggests that G4-binding ligands could be a critical tool in fighting infections. This review provides an overview of the progress and applications of G4-binding ligands, their proposed mechanisms of action, challenges faced and prospects for their utilization in anticancer treatments, neurological disorders and antiviral activities.
DNAzymes have emerged as an important class of sensors for a wide variety of metal ions, with florescence DNAzyme sensors as the most widely used in different sensing and imaging applications because of their fast response time, high signal intensity, and high sensitivity. However, the requirements of an external excitation light source and its associated power increase the cost and size of the fluorometer, making it difficult to be used for portable detections. To overcome these limitations, we report herein a DNAzyme sensor that relies on chemiluminescence resonance energy transfer (CRET) without the need for external light. The sensor is constructed by combining the functional motifs from both Pb2+-dependent 8-17 DNAzyme conjugated to fluorescein (FAM) and hemin/G-quadruplex that mimics horseradish peroxidase to catalyze the oxidation of luminol by H2O2 to yield chemiluminescence. In the absence of Pb2+, the hybridization between the enzyme and substrate strands bring the FAM and hemin/G-quadruplex in close proximity, resulting in CRET. The presence of Pb2+ ions can drive the cleavage on the substrate strand, resulting in a sharp decrease in the melting temperature of hybridization and thus separation of the FAM from hemin/G-quadruplex. The liberated CRET pair causes a ratiometric increase in the donor's fluorescent signal and a decrease in the acceptor signal. Using this method, Pb2+ ions have been measured rapidly (<15 min) with a low limit of detection at 5 nM. By removing the requirement of exogenous light excitation, we have demonstrated a simple and portable detection using a smartphone, making the DNAzyme-CRET system suitable for field tests of lake water. Since DNAzymes selective for other metal ions or targets, such as bacteria, can be obtained using in vitro selection, the method reported here opens a new avenue for rapid, portable, and ratiometric detection of many targets in environmental monitoring, food safety, and medical diagnostics.
G-quadruplexes are made up of guanine-rich RNA and DNA sequences capable of forming noncanonical nucleic acid secondary structures. The base-specific sterical configuration of G-quadruplexes allows the stacked G-tetrads to bind certain planar molecules like hemin (iron (III)-protoporphyrin IX) to regulate enzymatic-like functions such as peroxidase-mimicking activity, hence the use of the term DNAzyme/RNAzyme. This ability has been widely touted as a suitable substitute to conventional enzymatic reporter systems in diagnostics. This review will provide a brief overview of the G-quadruplex architecture as well as the many forms of reporter systems ranging from absorbance to luminescence readouts in various platforms. Furthermore, some challenges and improvements that have been introduced to improve the application of G-quadruplex in diagnostics will be highlighted. As the field of diagnostics has evolved to apply different detection systems, the need for alternative reporter systems such as G-quadruplexes is also paramount.
G-quadruplex (G4) ligands attract considerable attention as potential anticancer therapeutics. In this study we proposed an original scheme for synthesis of azole-fused anthraquinones and prepared a series of G4 ligands carrying amino- or guanidinoalkylamino side chains. The heterocyclic core and structure of the terminal groups strongly affect on binding to G4-forming oligonucleotides, cellular accumulation and antitumor potency of compounds. In particular, thiadiazole- and selenadiazole- but not triazole-based ligands inhibit the proliferation of tumor cells (e.g. K562 leukemia) and stabilize primarily telomeric and c-MYC G4s. Anthraselenadiazole derivative 11a showed a good affinity to c-MYC G4 in vitro and down-regulated expression of c-MYC oncogene in cellular conditions. Further studies revealed that anthraselenadiazole 11a provoked cell cycle arrest and apoptosis in a dose- and time-dependent manner inhibiting K562 cells growth. Taken together, this work gives a valuable example that the closely related heterocycles may cause a significant difference in biological properties of G4 ligands.
Immunoassays are often coupled to peroxidase activity for antigen detection. Sensitivity and speed of detection has been increased by the advent of hybrid methods such as immuno-PCR (polymerase chain reaction). However, a more simplified immunoassay that retains both colorimetric peroxidase detection and effective DNA amplification in a setting closer to field application conditions has been nonexistent. Here we describe a method that successfully combines a competitive immunoassay with the new isothermal quadruplex-primed amplification (QPA) to generate excess quadruplex reporter molecules with intrinsic peroxidase DNAzyme activity.
Next generation DNA sequencing and analysis of amplicons spanning the pharmacogene CYP2D6 suggested that the Nextera transposase used for fragmenting and providing sequencing priming sites displayed a targeting bias. This manifested as dramatically lower sequencing coverage at sites in the amplicon that appeared likely to form G-quadruplex structures. Since secondary DNA structures such as G-quadruplexes are abundant in the human genome, and are known to interact with many other proteins, we further investigated these sites of low coverage. Our investigation revealed that G-quadruplex structures are formed in vitro within the CYP2D6 pharmacogene at these sites, and G-quadruplexes can interact with the hyperactive Tn5 transposase (EZ-Tn5) with high affinity. These findings indicate that secondary DNA structures such as G-quadruplexes may represent preferential transposon integration sites and provide additional evidence for the role of G-quadruplex structures in transposition or viral integration processes.
The presence of heavy metal in food chains due to the rapid industrialization poses a serious threat on the environment. Therefore, detection and monitoring of heavy metals contamination are gaining more attention nowadays. However, the current analytical methods (based on spectroscopy) for the detection of heavy metal contamination are often very expensive, tedious and can only be handled by trained personnel. DNA biosensors, which are based on electrochemical transduction, is a sensitive but inexpensive method of detection. The principles, sensitivity, selectivity and challenges of electrochemical biosensors are discussed in this review. This review also highlights the major advances of DNA-based electrochemical biosensors for the detection of heavy metal ions such as Hg(2+), Ag(+), Cu(2+) and Pb(2+).
By inhibiting only two or three of 12 restriction enzymes, the series of [M(phen)(edda)] complexes [M(II) is Cu, Co, Zn; phen is 1,10-phenanthroline; edda is N,N'-ethylenediaminediacetate] exhibit DNA binding specificity. The Cu(II) and Zn(II) complexes could differentiate the palindromic sequences 5'-CATATG-3' and 5'-GTATAC-3', whereas the Co(II) analogue could not. This and other differences in their biological properties may arise from distinct differences in their octahedral structures. The complexes could inhibit topoisomerase I, stabilize or destabilize G-quadruplex, and lower the mitochondrial membrane potential of MCF7 breast cells. The pronounced stabilization of G-quadruplex by the Zn(II) complex may account for the additional ability of only the Zn(II) complex to induce cell cycle arrest in S phase. On the basis of the known action of anticancer compounds against the above-mentioned individual targets, we suggest the mode of action of the present complexes could involve multiple targets. Cytotoxicity studies with MCF10A and cisplatin-resistant MCF7 suggest that these complexes exhibit selectivity towards breast cancer cells over normal ones.