G-quadruplexes for luminescent sensing and logic gates.

G-quadruplexes represent a versatile sensing platform for the construction of label-free molecular detection assays owing to their diverse structures that can be selectively recognized by G-quadruplex-specific luminescent probes. In this Survey and Summary, we highlight recent examples of the application of the label-free strategy for the development of G-quadruplex-based luminescent detection platforms with a view towards the potential application of tetraplex structures in the design of DNA logic gates.

A label-free G-quadruplex-based luminescent switch-on assay for the selective detection of histidine.

A label-free G-quadruplex-based luminescent switch-on assay has been developed for the selective detection of micromolar histidine in aqueous solution. In this study, an iridium(III) complex was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5'-TG4AG3TG4AG3TG4A2G2-3')/cupric ion (Cu(2+)) ensemble was employed as a recognition unit for histidine. The initial luminescence of the iridium(III) complex in the presence of G-quadruplex DNA is effectively quenched by Cu(2+) ions due to the Cu(2+)-mediated unfolding of the G-quadruplex motif. The addition of histidine sequesters Cu(2+) ions from the ensemble, thereby restoring the luminescence of the system. The assay could detect down to 1 µM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine, masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated.

Detection of 3'→5' exonuclease activity using a metal-based luminescent switch-on probe.

A luminescent iridium(III) complex has been discovered to be selective for G-quadruplex DNA, and was employed in a label-free G-quadruplex-based detection assay for 3'→5' exonuclease activity in aqueous solution. A proof-of-concept of this assay has been demonstrated by using prokaryotic exonuclease III (ExoIII) as a model enzyme. In this assay, a G-quadruplex-forming hairpin oligonucleotide (hairpin-G4 DNA, 5'-GAG3TG4AG3TG4A2GCAGA2G2ATA2CT2C4AC3TC4AC3TC-3') initially exists in a duplex conformation, resulting in a low luminescence signal due to the weak interaction between the iridium(III) complex and duplex DNA. Upon digestion by ExoIII, the guanine-rich sequence is released and folds into a G-quadruplex, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for 3'→5' exonuclease over other DNA-modifying enzymes.

A parallel G-quadruplex-selective luminescent probe for the detection of nanomolar calcium(II) ion.

A parallel G-quadruplex-selective iridium(III) complex has been synthesized and employed as a luminescent probe in a label-free G-quadruplex-based detection assay for Ca(2+) ions in aqueous solution. In this assay, a guanine-rich oligonucleotide (G4, 5'-G4T4G4-3') initially exists in an antiparallel G-quadruplex conformation, resulting in a low luminescence signal. Upon incubation with Ca(2+) ions, the antiparallel G-quadruplex is induced into a parallel G-quadruplex conformation, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for Ca(2+) ions with a limit of detection in the nanomolar range, and was selective for Ca(2+) over other metal ions.

A highly sensitive G-quadruplex-based luminescent switch-on probe for the detection of polymerase 3'-5' proofreading activity.

We report herein a luminescent switch-on label-free G-quadruplex-based assay for the rapid and sensitive detection of polymerase proofreading activity using a novel iridium(III) complex as a G-quadruplex-selective probe. The interaction of the iridium(III) complex with the G-quadruplex motif facilitates the highly sensitive switch-on detection of polymerase proofreading activity. Using T4 DNA polymerase (T4 pol) as a model enzyme, the assay achieved high sensitivity and selectivity for T4 pol over other tested enzymes.

Luminescent detection of DNA-binding proteins.

Transcription factors play a central role in cell development, differentiation and growth in biological systems due to their ability to regulate gene expression by binding to specific DNA sequences within the nucleus. The dysregulation of transcription factor signaling has been implicated in the pathogenesis of a number of cancers, developmental disorders, inflammation and autoimmunity. There is thus a high demand for convenient high-throughput methodologies able to detect sequence-specific DNA-binding proteins and monitor their DNA-binding activities. Traditional approaches for protein detection include gel mobility shift assays, DNA footprinting and enzyme-linked immunosorbent assays (ELISAs) which tend to be tedious, time-consuming, and may necessitate the use of radiographic labeling. By contrast, luminescence technologies offer the potential for rapid, sensitive and low-cost detection that are amenable to high-throughput and real-time analysis. The discoveries of molecular beacons and aptamers have spear-headed the development of new luminescent methodologies for the detection of proteins over the last decade. We survey here recent advances in the development of luminescent detection methods for DNA-binding proteins, including those based on molecular beacons, aptamer beacons, label-free techniques and exonuclease protection.

Label-free luminescent oligonucleotide-based probes.

Breakthrough advances in chemistry and biology over the last two decades have vastly expanded the repertoire of nucleic acid structure and function with potential application in multiple areas of science and technology, including sensing and analytical applications. DNA oligonucleotides represent popular tools for the development of sensing platforms due to their low cost, rich structural polymorphism, and their ability to bind to cognate ligands with sensitivity and specificity rivaling those for protein enzymes and antibodies. In this review, we give an overview of the "label-free" approach that has been a particular focus of our group and others for the construction of luminescent DNA-based sensing platforms. The label-free strategy aims to overcome some of the drawbacks associated with the use of covalently-labeled oligonucleotides prevalent in electrochemical and optical platforms. Label-free DNA-based probes harness the selective interaction between luminescent dyes and functional oligonucleotides that exhibit a "structure-switching" response upon binding to analytes. Based on the numerous examples of label-free luminescent DNA-based probes reported recently, we envisage that this field would continue to thrive and mature in the years to come.

Bioactive luminescent transition-metal complexes for biomedical applications.

The serendipitous discovery of the anticancer drug cisplatin cemented medicinal inorganic chemistry as an independent discipline in the 1960s. Luminescent metal complexes have subsequently been widely applied for sensing, bio-imaging, and in organic light-emitting diode applications. Transition-metal complexes possess a variety of advantages that make them suitable as therapeutics and as luminescent probes for biomolecules. It is thus highly desirable to develop new luminescent metal complexes that either interact with DNA through different binding modes or target alternative cellular machinery such as proteins as well as to provide a more effective means of monitoring disease progression. In this Review, we highlight recent examples of biologically active luminescent metal complexes that can target and probe a specific biomolecule, and offer insights into the future potential of these compounds for the investigation and treatment of human diseases.

Combination of i-motif and G-quadruplex structures within the same strand: formation and application.

Peaceful coexistence: A double quadruplex composed of an i-motif and a G-quadruplex was constructed within one oligonucleotide strand (see picture). The defined double-quadruplex structure can serve as a NOTIF logic gate on the basis of the fluorescence of crystal violet.

Nanoscale coordination polymers enabling antioxidants inhibition for enhanced chemodynamic therapy.

Reactive oxygen species (ROS) generation to induce cell death is an effective strategy for cancer therapy. In particular, chemodynamic therapy (CDT), using Fenton-type reactions to generate highly cytotoxic hydroxyl radical (•OH), is a promising treatment modality. However, the therapeutic efficacy of ROS-based cancer treatment is still limited by some critical challenges, such as overexpression of enzymatic and non-enzymatic antioxidants by tumor cells, as well as the low tumor targeting efficiency of therapeutic agents. To address those problems, biomimetic CuZn protoporphyrin IX nanoscale coordination polymers have been developed, which significantly amplify oxidative stress against tumors by simultaneously inhibiting enzymatic and non-enzymatic antioxidants and initiating the CDT. In this design, cancer cell membrane camouflaged nanoparticle exhibits an excellent homotypic targeting effect. After being endocytosed into tumor cells, the nanoparticles induce depletion of the main non-enzymatic antioxidant glutathione (GSH) by undergoing a redox reaction with GSH. Afterward, the redox reaction generated cuprous ion (Cu+) works as a CDT agent for •OH generation. Furthermore, the released Zn protoporphyrin IX strongly inhibits the activity of the typical enzymatic antioxidant heme oxygenase-1. This tetra-modal synergistic strategy endows the biomimetic nanoparticles with great capability for anticancer therapy, which has been demonstrated in both in vitro and in vivo studies.

A label-free G-quadruplex-based switch-on fluorescence assay for the selective detection of ATP.

A G-quadruplex-based, label-free, switch-on fluorescence detection method has been developed for the selective detection of ATP in aqueous solution using crystal violet as a G-quadruplex-selective probe. The assay is highly simple and rapid, and does not require the use of fluorescent labeling.

An Ultrafast One-Step Quantitative Reverse Transcription-Polymerase Chain Reaction Assay for Detection of SARS-CoV-2.

We developed an ultrafast one-step RT-qPCR assay for SARS-CoV-2 detection, which can be completed in only 30 min on benchtop Bio-Rad CFX96. The assay significantly reduces the running time of conventional RT-qPCR: reduced RT step from 10 to 1 min, and reduced the PCR cycle of denaturation from 10 to 1 s and extension from 30 to 1 s. A cohort of 60 nasopharyngeal swab samples testing showed that the assay had a clinical sensitivity of 100% and a clinical specificity of 100%.

High-throughput negative detection of SDS-PAGE separated proteins and its application for proteomics.

A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, low-cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.

A user-friendly alternative to formaldehyde-based DNA silver-staining method on polyacrylamide gels.

We have developed a practical, cost-effective and user-friendly protocol to meet the needs of nucleic acids research, particularly in respect of DNA detection on polyacrylamide gels. In this method, the most commonly used alkaline formaldehyde developer in DNA silver stain, which does harm to operator, is first replaced by glucose in alkaline borate buffer. In addition, the effects of six reducing sugars on the quality of DNA visualization were investigated. Consequently, the optimal protocol using glucose takes about 45 min to complete all the procedures, with a detection limit of 5 pg of single DNA band on polyacrylamide gels, was developed. The results indicate that this user-friendly and economic protocol could be a good choice for routine use in DNA visualization on polyacrylamide gels.

Improved conditions for silver-ammonia staining of DNA in polyacrylamide gel.

An improved silver-ammonia staining method for DNA on polyacrylamide gels is described. In this method, staining of DNA using silver-ammonia complex allows high sensitivity, low cost, low toxicity, and simple protocol without requiring fixation and sensitization steps. The protocol takes less than 40 min to complete, with a detection limit of 1.5 pg of single DNA band on polyacrylamide gels, approximately 30-fold higher than that of original silver-ammonia staining method. Furthermore, this novel technique not only exhibits high sensitivity for large DNA fragment, but also shows a better trend to detect low-base-pair DNA compared with other silver staining methods.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y.

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.

We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain.

Extracellular Vesicles as Potential Biomarkers for Early Detection and Diagnosis of Pancreatic Cancer.

Pancreatic carcinoma (PC) is highly metastatic, and it tends to be detected at advanced stages. Identifying and developing biomarkers for early detection of PC is crucial for a potentially curative treatment. Extracellular vesicles (EVs) are bilayer lipid membrane-structured nanovesicles found in various human bodily fluids, and they play important roles in tumor biogenesis and metastasis. Cancer-derived EVs are enriched with DNA, RNA, protein, and lipid, and they have emerged as attractive diagnostic biomarkers for early detection of PC. In this article, we provided an overview of the cell biology of EVs and their isolation and analysis, and their roles in cancer pathogenesis and progression. Multiplatform analyses of plasma-based exosomes for genomic DNA, micro RNA, mRNA, circular RNA, and protein for diagnosis of PC were critically reviewed. Numerous lines of evidence demonstrate that liquid biopsy with analysis of EV-based biomarkers has variable performance for diagnosis of PC. Future investigation is indicated to optimize the methodology for isolating and analyzing EVs and to identify the combination of EV-based biomarkers and other clinical datasets, with the goal of improving the predictive value, sensitivity, and specificity of screening tests for early detection and diagnosis of PC.

Enrichment of extracellular vesicles with lipid nanoprobe functionalized nanostructured silica.

Nanoscale extracellular vesicles (nEVs) have recently demonstrated potential value in cancer diagnostics and treatment monitoring, but translation has been limited by technical challenges in nEV isolation. Thus, we have developed a one-step nEV isolation platform that utilizes nEV size-matched silica nanostructures and a surface-conjugated lipid nanoprobe with an integrated microfluidic mixer. The reported platform has 28.8% capture efficiency from pancreatic cancer plasma and can sufficiently enrich nEVs for simpler positive identification of point mutations, particularly KRAS, in nEV DNA from the plasma of pancreatic cancer patients.

Label-free luminescence switch-on detection of hepatitis C virus NS3 helicase activity using a G-quadruplex-selective probe.

A series of luminescent Ir(iii) complexes were synthesised and evaluated for their ability to act as luminescent G-quadruplex-selective probes. The Ir(iii) complex 9, [Ir(phq)2(phen)]PF6 (where phq = 2-phenylquinoline; phen = 1,10-phenanthroline), exhibited high luminescence in the presence of G-quadruplex DNA compared to dsDNA and ssDNA, and was employed to construct a label-free G-quadruplex-based assay for hepatitis C virus NS3 helicase activity in aqueous solution. Moreover, the application of the assay for screening potential helicase inhibitors was demonstrated. To our knowledge, this is the first G-quadruplex-based assay for helicase activity.