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Abstract: Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1, for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes. [ABSTRACT FROM AUTHOR]

Abstract: Because of the absence of methods for tracking RNA G‐quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G‐quadruplexes is so far limited. Herein, we report a new red‐emitting fluorescent probe, QUMA‐1, for the selective, continuous, and real‐time visualization of RNA G‐quadruplexes in live cells. The applications of QUMA‐1 in several previously intractable applications, including live‐cell imaging of the dynamic folding, unfolding, and movement of RNA G‐quadruplexes and the visualization of the unwinding of RNA G‐quadruplexes by RNA helicase have been demonstrated. Notably, our real‐time results revealed the complexity of the dynamics of RNA G‐quadruplexes in live cells. We anticipate that the further application of QUMA‐1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G‐quadruplexes will uncover more information about the biological roles of RNA G‐quadruplexes. [ABSTRACT FROM AUTHOR]

Helicases are crucial enzymes in DNA and RNA metabolism and function by unwinding particular nucleic acid structures. However, most convenient and high-throughput helicase assays are limited to the typical duplex DNA. Herein, we developed an immunosorbent assay to monitor the Werner syndrome (WRN) helicase unwinding a wide range of DNA structures, such as a replication fork, a bubble, Holliday junction, G-quadruplex and hairpin. This assay could sensitively detect the unwinding of DNA structures with detection limits around 0.1 nM, and accurately monitor the substrate-specificity of WRN with a comparatively less time-consuming and high throughput process. Remarkably, we have established that this new assay was compatible in evaluating helicase inhibitors and revealed that the inhibitory effect was substrate-dependent, suggesting that diverse substrate structures other than duplex structures should be considered in discovering new inhibitors. Our study provided a foundational example for using this new assay as a powerful tool to study helicase functions and discover potent inhibitors. [ABSTRACT FROM AUTHOR]

Mitochondria have a crucial role in regulating energy metabolism and their dysfunction has been linked to tumorigenesis. Cancer diagnosis and intervention have a great interest in the development of new agents that target biomolecules within mitochondria. However, monitoring and modulating mitochondria RNA (mtRNA), an essential component in mitochondria, in cells is challenging due to limited functional research and the absence of targeting agents. In this study, we designed and synthesized a fluorescent quinolinium derivative, QUCO-1, which actively lit up with mtRNA in both normal and cancer cells in vitro. Additionally, we evaluated the function of QUCO-1 as an mtRNA ligand and found that it effectively induced severe mitochondrial dysfunction and OXPHOS inhibition in RKO colorectal cancer cells. Treatment with QUCO-1 resulted in apoptosis, cell cycle blockage at the G2/M phase, and the effective inhibition of cell proliferation. Our findings suggest that QUCO-1 has great potential as a promising probe and therapeutic agent for mtRNA, with the potential for treating colorectal cancer. [ABSTRACT FROM AUTHOR]

RNA imaging is of great importance for understanding its complex spatiotemporal dynamics and cellular functions. Considerable effort has been devoted to the development of small-molecule fluorescent probes for RNA imaging. However, most of the reported studies have mainly focused on improving the photostability, permeability, long emission wavelength, and compatibility with live-cell imaging of RNA probes. Less attention has been paid to the selectivity and detection limit of this class of probes. Highly selective and sensitive RNA probes are still rarely available. In this study, a new set of styryl probes were designed and synthesized, with the aim of upgrading the detection limit and maintaining the selectivity of a lead probe QUID−1 for RNA. Among these newly synthesized compounds, QUID−2 was the most promising candidate. The limit of detection (LOD) value of QUID−2 for the RNA was up to 1.8 ng/mL in solution. This property was significantly improved in comparison with that of QUID−1. Further spectroscopy and cell imaging studies demonstrated the advantages of QUID−2 over a commercially available RNA staining probe, SYTO RNASelect, for highly selective and sensitive RNA imaging. In addition, QUID−2 exhibited excellent photostability and low cytotoxicity. Using QUID−2, the global dynamics of RNA were revealed in live cells. More importantly, QUID−2 was found to be potentially applicable for detecting RNA granules in live cells. Collectively, our work provides an ideal probe for RNA imaging. We anticipate that this powerful tool may create new opportunities to investigate the underlying roles of RNA and RNA granules in live cells. [ABSTRACT FROM AUTHOR]

• A new strategy was developed to discover RNA G-quadruplex-specific fluorescent probe. • The BEDO-3 is the first green-emitting RNA G-quadruplex-specific fluorescent probe. • Our findings will illuminate the development of brand-new RNA G-quadruplex probes. The visualization of RNA G-quadruplexes in cells is critical to studying and understanding their biological functions. However, while various fluorescent probes have been developed for RNA imaging, very few RNA G-quadruplex-specific fluorescent probes have been found. To address this challenge, we reasoned that turning an RNA fluorescent probe into an RNA G-quadruplex-specific fluorescent probe by rational structural modification to improve the specificity would be an efficient new strategy. Therefore, the benzothiazole-indole-based RNA fluorescent probe BEDO-0 was selected to test this approach. Based on its sensing mechanism, we designed several candidate probes with the aim of improving their G-quadruplex binding selectivity while maintaining the specific fluorescence response. Among them, BEDO-3 , which possesses an extended aromatic ring and amino side chain targeting the G-quartet and phosphate backbone, is the best candidate with the strongest G-quadruplex binding affinity and specificity. The relationships between the candidate structure and imaging performance were examined in detail. Furthermore, the practicability of using BEDO-3 as an RNA G-quadruplex-specific fluorescent probe was determined. These findings fully validate the feasibility of our proposed efficient and rational strategy, which will illuminate the development of brand-new fluorescent probes for RNA G-quadruplexes and even for other RNAs. [ABSTRACT FROM AUTHOR]

Stress granules (SGs) are a fascinating type of membrane-less organelle that form through liquid-liquid phase separation in eukaryotic cells. Our previous investigations have been instrumental in the development of the first small-molecule fluorescent probe, named TASG , which can selectively identify SGs. However, the practical utility of TASG for monitoring SG dynamics in live cells has been hindered by the difficulty in visualizing small nascent SGs. To overcome this obstacle, the present study systematically modified the structure of TASG , with the aim of discovering fluorescent chemical probes that exhibit superior performance. Among the evaluated candidates, TASG-8 exhibited the most promising ability to image SGs. Importantly, TASG-8 possessed a critical advantage over TASG , in that it could effectively label small nascent SGs. The structure-activity relationship of the candidates was analyzed. Additionally, the underlying mechanism responsible for this enhancement was extensively explored. Collectively, our research presents a rational strategy for the development of innovative fluorescent probes that selectively target SGs, and introduces a powerful new tool, TASG-8 , which has the potential to facilitate more comprehensive and efficient investigations into the functions of SGs in cells. • The structure-activity relationship of benzothiazoles that target SGs is clarified. • TASG-8 is the first fluorescent probe capable of visualizing small nascent SGs. • Our study presents a novel concept for developing probes that are specific to SGs. [ABSTRACT FROM AUTHOR]

[Display omitted] • SEMA-1 is the first benzoselenazolium-based hemicyanine for G-quadruplex detection. • SEMA-1 has potential to detect nucleolar and mitochondrial G-quadruplexes in cells. • Benzoselenazolium-based hemicyanine is a valuable scaffold for probe design. Benzothiazolium and benzoxazolium are common groups for the construction of hemicyanine dyes; however, their isosteric analogue benzoselenazolium have rarely been studied. Here, we report the development of the first benzoselenazolium-based hemicyanine dye for the selective detection of G-quadruplexes. This molecule, SEMA-1 , was validated as a red-emitting and activatable fluorescent probe whose fluorescence would only be activated in the presence of G-quadruplexes in buffer solution. Consistent with this, SEMA-1 was found to accumulate in nucleoli and could be used to detect the high abundance of nucleolar rDNA and rRNA G-quadruplexes in fixed HeLa cells. On the other hand, due to the retained mitochondrial membrane potential in live HeLa cells, SEMA-1 was captured by mitochondria and had the potential to detect the mitochondrial G-quadruplexes. Collectively, this work demonstrates the value of developing G-quadruplex-specific fluorescent probes from novel benzoselenazolium-based hemicyanine scaffold. [ABSTRACT FROM AUTHOR]

Since many of the pivotal questions confronting chemical biology concern the interaction of more than one analyte, it is essential to investigate multiple analytes simultaneously. While commercial fluorescent dyes capable of detecting and quantitating DNA or RNA are widely used in biological research, few probes can be applied in DNA and RNA together. Distraction from each other is also a puzzling question for individual detection. Here, we have discovered a dual-responsive probe, namely, DR1 , which can selectively recognize DNA by emitting out red fluorescence in the nucleus, while an entirely different stain pattern by labeling cytoplasm and nuclei with distinct cyanine fluorescence is observed owing to binding with RNA. Mechanism study found that DR1 binds to DNA in a monomer state with red fluorescence, and therefore the cyanine fluorescence may derive from its aggregated state while it binds to RNA. Then, DR1 was applied to quantify DNA and RNA contents and evaluate the effects of several inhibitors comprehensively on high-content screening platforms. Our study provided a new tool for simultaneously monitoring cellular DNA and RNA, and gaining new insights into developing dual-channel probes for multiple biomolecules. • The dual-color imaging probe DR1 was developed to monitor DNA and RNA simultaneously in cells. • Dual-responsive mechanism of DR1 was discovered to be related to aggregation and monomer status. • A high-content imaging method of evaluating DNA and RNA modulators effects was established based on our probe. [ABSTRACT FROM AUTHOR]