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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]

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]