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]

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]