Search

G-quadruplexes (G4s) are peculiar nucleic acid secondary structures formed by DNA or RNA and are considered as fundamental features of the genome. Many proteins can specifically bind to G4 structures. There is increasing evidence that G4–protein interactions involve in the regulation of important cellular processes, such as DNA replication, transcription, RNA splicing, and translation. Additionally, G4–protein interactions have been demonstrated to be potential targets for disease treatment. In order to unravel the detailed regulatory mechanisms of G4-binding proteins (G4BPs), biochemical methods for detecting G4–protein interactions with high specificity and sensitivity are highly demanded. Here, we review recent advances in screening and validation of new G4BPs and highlight both their features and limitations. Affinity enrichment methods and G-quadruplex (G4) ligand-mediated photo-crosslinking methods can be used to screen new G4-binding proteins (G4BPs) with high throughput and accuracy. Many in vitro methods based on classic nucleic acid–protein interaction assays have been developed to validate G4BPs, but methods for investigating G4–protein interactions within the cellular milieu are still under demand. The development of more highly sensitive, selective, and high-throughput biochemical methods will greatly facilitate the development of G4BP biology. [ABSTRACT FROM AUTHOR]

G‐quadruplex (G4) structures exist in the single‐stranded DNA of chromatin and regulate genome function. However, the native chromatin G4 landscape in living cells has yet to be fully characterized. Herein, a genetic‐encoded live‐cell G4 identifier probe (LiveG4ID) is constructed and its cellular localization, biocompatibility, and G4‐binding specificity is evaluated. By coupling LiveG4ID with cleavage under targets and tagmentation (CUT&Tag), LiveG4ID‐seq, a method for mapping native chromatin G4 landscape in living cells with high accuracy is established. Compared to the conventional G4 CUT&Tag method, LiveG4ID‐seq can identify more chromatin G4 signals and have a higher ratio of true positive signals. Using LiveG4ID‐seq, the dynamic landscape of chromatin G4 structures during the cell cycle is profiled. It is discovered that chromatin G4 structures are prevalent in the promoter regions of cell cycle‐specific genes, even in the early M phase when the chromatin is condensed. These data demonstrate the capacity of LiveG4ID‐seq to profile a more accurate G4 landscape in living cells and promote future studies on chromatin G4 structures. [ABSTRACT FROM AUTHOR]

Single‐cell imaging has unique advantages of maintaining the in situ physiological state, morphology, and microenvironment, becoming a powerful tool to unravel the nature of intracellular nucleic acids. The analysis of nucleic acids unprecedentedly demands the sub‐molecule details at segment or subunit, secondary structure and monomer levels, instead of just probing the sequence and the abundance of nucleic acids. Detection of nucleic acids at the sub‐molecule level requires higher specificity and higher sensitivity, which becomes a new challenge in nucleic acid analysis. Herein, we summarize the recent progress in the design and the application of single‐cell nucleic acid imaging methods at the sub‐molecule level, including the visualization of RNA splicing variants, RNA G‐quadruplexes in an individual gene, single nucleotide variation of mitochondrial DNA, and RNA m6A methylation. Remarkably, we highlight the key strategy, "Module Assembly", for high‐performance molecular recognition and demonstrate the required improvements in future research. [ABSTRACT FROM AUTHOR]

G‐quadruplexes (G4s), non‐canonical nucleic acid secondary structure, regulate many biological functions and are considered potential molecular targets for cancer therapeutics. However, due to the lack of analytical methods, the regulating mechanism of monogenic G4s is still unclear. Here, we developed a Module Assembled Multifunctional Probes Assay (MAMPA) for visualizing endogenous G4s in individual genes in single cells. Two modular probes separately recognize G4 structures and the adjacent RNA sequences, and the module assembly enables imaging of G4s in an individual RNA with high specificity. Through imaging G4s in several individual genes, we found that G4s were steadily occupied by G4 Binding Proteins (G4BPs) in various mRNAs in every cell line and defined "Occupied G4 Ratio". We demonstrated MAMPA was suitable for most experimental situations and found that Occupied G4 Ratios had the potential to become a new parameter for the study of G4s in living cells. [ABSTRACT FROM AUTHOR]

G‐quadruplexes (G4s), non‐canonical nucleic acid secondary structure, regulate many biological functions and are considered potential molecular targets for cancer therapeutics. However, due to the lack of analytical methods, the regulating mechanism of monogenic G4s is still unclear. Here, we developed a Module Assembled Multifunctional Probes Assay (MAMPA) for visualizing endogenous G4s in individual genes in single cells. Two modular probes separately recognize G4 structures and the adjacent RNA sequences, and the module assembly enables imaging of G4s in an individual RNA with high specificity. Through imaging G4s in several individual genes, we found that G4s were steadily occupied by G4 Binding Proteins (G4BPs) in various mRNAs in every cell line and defined "Occupied G4 Ratio". We demonstrated MAMPA was suitable for most experimental situations and found that Occupied G4 Ratios had the potential to become a new parameter for the study of G4s in living cells. [ABSTRACT FROM AUTHOR]

Glioblastoma (GBM) is the most malignant brain tumor with unmet therapeutic demand. The blood‐brain‐barrier (BBB) and tumor heterogeneity limit the treatment effectiveness of various interventions. Here, an ultrasound augmented chemo/immuno therapy for GBM using a neutrophil‐delivered nanosensitizer, is developed. The sensitizer is composed of a ZnGa2O4:Cr3+ (ZGO) core for persistent luminescence imaging and a hollow sono‐sensitive TiO2 shell to generate reactive oxygen species (ROS) for controlled drug release. Immune checkpoint inhibitor (Anti‐PD‐1 antibody) is trapped in the interior of the porous ZGO@TiO2 with paclitaxel (PTX) loaded liposome encapsulation to form ZGO@TiO2@ALP. Delivered by neutrophils (NEs), ZGO@TiO2@ALP‐NEs can penetrate through BBB for GBM accumulation. After intravenous injection, ultrasound irradiation at GBM sites initiates ROS generation from ZGO@TiO2@ALP, leading to liposome destruction for PTX and anti‐PD‐1 antibody release to kill tumors and induce local inflammation, which in‐turn attractes more ZGO@TiO2@ALP‐NEs to migrate into tumor sites for augmented and sustained therapy. The treatment enhances the survival rate of the GBM bearing mice from 0% to 40% and endows them with long‐term immuno‐surveillance for tumor recurrence, providing a new approach for precision therapy against GBM and other cancers. [ABSTRACT FROM AUTHOR]

Stroke is a lethal cerebral disease with severe sequelae and high mortality. Microglia, the main immune cell in the cerebrum, possess therapeutic potential for strokes as its specific anti‐inflammatory phenotype can reduce inflammation and promote neuron regeneration. However, the on‐demand anti‐inflammatory polarization of microglia at the stroke site is uncontrollable for therapeutic application. Here, we develop a platelet hybrid microglia platform which can specifically polarize to the anti‐inflammatory phenotype by ultrasound irradiation for targeted cerebrum repair after stroke. The engineered microglia have strong adherence to the injured cerebral vessels with platelet membrane fusion and realize on‐demand anti‐inflammatory polarization with ultrasound‐responsive IL‐4 liposome decoration. The intravenously injected microglia platform showed anti‐inflammatory polarization at the stroke site with insonation, and accelerated the M2‐type polarization of endogenous microglia for long‐term stroke recovery. Satisfied prognoses were achieved with reduced apoptosis, promoted neurogenesis, and functional recovery, indicating the implications of the microglia platform for stroke therapy. [ABSTRACT FROM AUTHOR]

Stroke is a lethal cerebral disease with severe sequelae and high mortality. Microglia, the main immune cell in the cerebrum, possess therapeutic potential for strokes as its specific anti‐inflammatory phenotype can reduce inflammation and promote neuron regeneration. However, the on‐demand anti‐inflammatory polarization of microglia at the stroke site is uncontrollable for therapeutic application. Here, we develop a platelet hybrid microglia platform which can specifically polarize to the anti‐inflammatory phenotype by ultrasound irradiation for targeted cerebrum repair after stroke. The engineered microglia have strong adherence to the injured cerebral vessels with platelet membrane fusion and realize on‐demand anti‐inflammatory polarization with ultrasound‐responsive IL‐4 liposome decoration. The intravenously injected microglia platform showed anti‐inflammatory polarization at the stroke site with insonation, and accelerated the M2‐type polarization of endogenous microglia for long‐term stroke recovery. Satisfied prognoses were achieved with reduced apoptosis, promoted neurogenesis, and functional recovery, indicating the implications of the microglia platform for stroke therapy. [ABSTRACT FROM AUTHOR]

Imaging agents, molecular recognition, nucleic acids, single-cell imaging, sub-molecule level Frontispiece: Module Assembly Strategy for Single-Cell Nucleic Acid Imaging at the Sub-Molecule Level Here, the key strategy, "Module Assembly", for single-cell imaging of nucleic acids at the sub-molecule level is reviewed. [Extracted from the article]

Pathogenic infection remains the primary threat to human health, such as the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutated variants, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we firstly describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges involved in on-site diagnostic assays and present emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics. • Class 2 and recently explored class 1 CRISPR tools are presented. • POCT strategies for detecting pathogenic viruses, bacteria and fungi are introduced. • CRISPR-based profiling of viral variants and drug-resistant bacteria is highlighted. • Challenges for multiplexing and preamplification-free diagnostic assay are discussed. [ABSTRACT FROM AUTHOR]

Single cell analysis is of great significance to biological and biomedical research. With recent development, lots of in situ amplification methods have been applied for imaging the spatial location of specific biomolecules in situ in single cells. In particular, RCA is a simple and efficient isothermal amplification method, which can locally amplify the fluorescent signal in situ. Due to the ability of detecting single molecules directly in cells and tissues, RCA has been widely used for illustrating many important biological processes. In this review, we summarize recent development of RCA-based method for single cell analysis and discuss their applications. Moreover, we emphasize the utilization of RCA for in situ sequencing, which is a very interesting and emerging research field. • The basic mechanism of rolling circle amplification (RCA) for in situ single cell imaging is summarized. • RCA can be used for analysis of various targets in situ in single cells, including gene expression, single-nucleotide variation, mRNA splicing and protein post-translation modification. • The utilization of RCA for in situ sequencing are discussed. [ABSTRACT FROM AUTHOR]

The ability to 'see' genetic information directly in single cells can provide invaluable insights into complex biological systems. In this review, we discuss recent advances of in situ imaging technologies for visualizing the subtlest sequence alteration, single-nucleotide variation (SNV), at single-cell level. The mechanism of recently developed methods for SNV discrimination are summarized in detail. With recent developments, single-cell SNV imaging methods have opened a new door for studying the heterogenous and stochastic genetic information in individual cells. Furthermore, SNV imaging can be used on morphologically preserved tissue, which can provide information on histological context for gene expression profiling in basic research and genetic diagnosis. Moreover, the ability to visualize SNVs in situ can be further developed into in situ sequencing technology. We expect this review to inspire more research work into in situ SNV imaging technologies for investigating cellular phenotypes and gene regulation at single-nucleotide resolution, and developing new clinical and biomedical applications. [ABSTRACT FROM AUTHOR]