Real-time monitoring of DNA G-quadruplexes in living cells with a small-molecule fluorescent probe.

G-quadruplex DNA has been viewed as a prospective anti-cancer target owing to its potential biological relevance. Real-time monitoring of DNA G-quadruplex structures in living cells can provide valuable insights into the relationship between G-quadruplex formation and its cellular consequences. However, the probes capable of detecting DNA G-quadruplexes in living cells are still very limited. Herein, we reported a new fluorescent probe, IMT, for real-time visualization of DNA G-quadruplex structures in living cells. Using IMT as a fluorescent indicator, the quantity changes of DNA G-quadruplex at different points in time during continuous cellular progression responding to Aphidicolin and Hydroxyurea treatment have been directly visualized. Our data demonstrate that IMT will be a valuable tool for exploring DNA G-quadruplexes in live cells. Further application of IMT in fluorescence imaging may reveal more information on the roles of DNA G-quadruplexes in biological systems.

A traceable nanoplatform for enhanced chemo-photodynamic therapy by reducing oxygen consumption.

Tumor hypoxia impedes the efficiencies of oxygen-dependent photodynamic therapy (PDT) and chemotherapy. Herein, we design a traceable nanoplatform (DOX/Met/BSA-HA-CDs) by reducing oxygen (O2) consumption to overcome the hypoxia-caused cancer therapy. Carbon dots (CDs) are used not only as a PDT agent but also applied for in vivo traceable imaging. Metformin (Met), a potent antihyperglycemic agent, to improve tumor oxygenation and enhance the efficiencies of hypoxia-caused cancer therapy. In the hypoxic tumor microenvironment, Met was released more rapidly than DOX, which is advantageous for improving hypoxic cancer to exert a better therapeutic efficiency. Ex vivo immunofluorescence staining revealed that the DOX/Met/BSA-HA-CDs nanoparticles greatly reduce O2 consumption in tumor site. Followed by in vivo synergistic treatment achieved considerably enhanced cancer therapeutic efficiency. This system holds great clinical promise as a traceable imaging approach to guide the improvement of PDT and chemotherapy efficiencies through utilizing a simple, safe method improved hypoxic tumor microenvironment.

Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe.

BACKGROUND: Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. METHODS: Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). RESULTS: Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. CONCLUSIONS: The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. GENERAL SIGNIFICANCE: The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells.

Aptamer-based plasmonic sensor array for discrimination of proteins and cells with the naked eye.

We developed a colorimetric sensor array with reported protein aptamers as nonspecific receptors. We found that different target proteins could make the aptamer-protected gold nanoparticles (AuNPs) exhibit different aggregation behaviors in the presence of a high concentration salt and cause various color change. On the basis of this phenomenon, we applied a series of reported protein aptamers as a receptor array obtaining a distinct response pattern to each target protein. Seven proteins have been well distinguished with the naked eye at the 50 nM level. Cancerous human cells have also been discriminated from noncancerous cells. This method is simple, label-free, and sensitive. It will broaden the application filed of plasmonic nanoparticle-based sensors and give a new direction of developing sensitive array sensing systems.

Monitoring autophagy in live cells with a fluorescent light-up probe for G-quadruplex structures.

Monitoring autophagy can provide valuable insights into understanding human pathological mechanisms, developing novel drugs, and exploring autophagy control approaches. Here, we proposed a new strategy to specifically monitor autophagy by lighting up the G-quadruplex structures entering autolysosomes. Based on this strategy, we designed a small-molecule fluorescent probe for autophagy imaging. This work not only opens up a new way for developing autophagy probes, but also provides an effective tool for autophagy research.

Evaluation of the selectivity of G-quadruplex ligands in living cells with a small molecule fluorescent probe.

G-quadruplex has been an emerging target for drug design due to its physiologically important roles in oncology. A number of quadruplex-interactive ligands have been developed by synthetic and medicinal chemists over the past decades. However, the great challenge still remains that the method for detecting the specific targeting of these ligands to the G-quadruplex structures in cells is still lacking. Herein, a detection system for directly identifying the specific targeting of a ligand to DNA G-quadruplexes in cells was constructed by using a small-molecular fluorescent probe (IMT) as a fluorescent indicator. Four typical ligands have been successfully evaluated, demonstrating the promising application of this detection system in the screening and evaluation of quadruplex-specific therapeutic agents.

Insulin-like growth factor type I selectively binds to G-quadruplex structures.

BACKGROUND: G-quadruplex has been viewed as a promising therapeutic target in oncology due to its potentially important roles in physiological and pathological processes. Emerging evidence suggests that the biological functions of G-quadruplexes are closely related to the binding of some proteins. Insulin-like growth factor type I (IGF-1), as a significant modulator of cell growth and development, may serve as a quadruplex-binding protein. METHODS: The binding affinity and selectivity of IGF-1 to different DNA motifs in solution were measured by using fluorescence spectroscopy, Surface Plasmon Resonance (SPR), and force-induced remnant magnetization (FIRM). The effects of IGF-1 on the formation and stability of G-quadruplex structures were evaluated by circular dichroism (CD) and melting fluorescence resonance energy transfer (FRET) spectroscopy. The influence of quadruplex-specific ligands on the binding of G-quadruplexes with IGF-1 was determined by FIRM. RESULTS: IGF-1 shows a binding specificity for G-quadruplex structures, especially the G-quadruplex structure with a parallel topology. The quadruplex-specific ligands TMPyP4 and PDS (Pyridostatin) can inhibit the interaction between G-quadruplexes and proteins. CONCLUSIONS: IGF-1 is demonstrated to selectively bind with G-quadruplex structures. The use of quadruplex-interactive ligands could modulate the binding of IGF-1 to G-quadruplexes. GENERAL SIGNIFICANCE: This study provides us with a new perspective to understand the possible physiological relationship between IGF-1 and G-quadruplexes and also conveys a strategy to regulate the interaction between G-quadruplex DNA and proteins.

Dual-functional carbon dot-labeled heavy-chain ferritin for self-targeting bio-imaging and chemo-photodynamic therapy.

Image-guided cancer nanotheranostics with a simple nano-platform are seriously significant for nanomedicine. In this study, a novel design is described to achieve sensitive bio-imaging and effective treatment by utilizing heavy-chain ferritin (HFn) nanocages as a vector coupled with dual-functional carbon dots (CDs) on the surface of ferritin and encapsulating the chemotherapeutic drug doxorubicin (DOX). The CDs obtained herein emit bright fluorescence in the red region, which can be applied to bio-imaging in vivo. More significantly, the CDs can produce reactive oxygen species (ROS) under laser irradiation at 532 nm and cause damage to the DNA in the nucleus. These unique properties enabled CDs to act as a theranostic agent. Owing to the self-targeting ability of HFn, the final nanoparticles can internalize into cancer cells more efficiently. The nanoparticles can translocate into the nucleus after DNA damage resulting from the partial release of DOX into the cytoplasm, thereby increasing the nuclear delivery of the drug. The results of this study indicate that the multifunctional HFn(DOX)/CD nanoparticles have potential as a clinically available cancer theranostic agent to deliver diagnostic agents and therapeutic drugs into the cancer cells and thus provide a noninvasive, highly sensitive imaging approach and guidance to the chemo-photodynamic therapy simultaneously.

Reversible regulation of the supramolecular chirality of a cyanine dye by using the G-quadruplex structure as a template.

Multiple cycle regulation of the supramolecular chirality of a cyanine dye has been successfully achieved by using DNA G-quadruplexes as templates, which is easily controllable by repeated addition of Ag(+) and cysteine (Cys). This work provides an easy and controllable strategy for the chiral regulation of supramolecules.