A Cytoplasm‐Specific Fluorescent Ligand for Selective Imaging of RNA G‐Quadruplexes in Live Cancer Cells

Zheng, Bo-Xin; Long, Wei; She, Meng‐Ting; Wang, Yakun; Zhao, Dong; Yu, Jie; Leung, Alan Siu-Lun; Chan, Ka Hin; Hou, Jinqiang; Lu, Yu‐Jing; Wong, Wing‐Leung

doi:10.1002/chem.202300705

Cited by 7 publications

(5 citation statements)

References 54 publications

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“…This preferential colocalization of TOR-G4 with RNA makes the molecule one of a limited number of probes well suited for studying G4 RNA within cells using fluorescence microscopy. 41–44…”

Section: Resultsmentioning

confidence: 99%

“…58 In comparison, visualization of RNA G4s (rG4s) is still a relatively new area of study. 41–44 Previously, it was suggested that RNA G4s may even be globally unfolded within cells due to the persistent activity of G4 helicases. 59 However, more recent studies have provided extensive evidence that rG4s not only exist within cells but may be pivotal for mediating RNA interactions with proteins that control processes such as splicing, translation and stress granule formation.…”

Section: Discussionmentioning

confidence: 99%

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Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

Robinson

Stenspil

Maleckaitė

et al. 2023

Preprint

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Over the last decade, appreciation of the potential roles of G-quadruplex (G4) structures in cellular regulation and maintenance have rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro, however, achieving sufficient selectivity to detect G4s with confidence in a dense and structurally diverse cellular environment is challenging. The use of fluorescence probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G-quadruplexes that does not rely on fluorescence intensity switch-on and thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independently of local probe concentration. Furthermore, TOR-G4 primarily co-localizes with RNA in the cytoplasm and nucleoli of cells, making it the first fluorescence lifetime-based probe validated for exploring the emerging roles of RNA G-quadruplexes in cellulo.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

Robinson

Stenspil

Maleckaitė

et al. 2023

Preprint

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“…The integration of molecular scaffolds was achieved via a rigid but rotatable ethylene bridge. The two π-conjugated scaffolds may form a coplanar structure upon interaction with targeted G4s and then the adduct can generate intensive fluorescence for real-time imaging and monitoring G4s in living cells . To the best of our knowledge, there are no such small-sized, dicationic, mitochondria-selective and G4-targeting ligands reported.…”

Section: Resultsmentioning

confidence: 99%

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy

Zheng,

Long,

Zheng

et al. 2024

J. Med. Chem.

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Mitochondria are important drug targets for anticancer and other disease therapies. Certain human mitochondrial DNA sequences capable of forming G-quadruplex structures (G4s) are emerging drug targets of small molecules. Despite some mitochondria-selective ligands being reported for drug delivery against cancers, the ligand design is mostly limited to the triphenylphosphonium scaffold. The ligand designed with lipophilic small-sized scaffolds bearing multipositive charges targeting the unique feature of high mitochondrial membrane potential (MMP) is lacking and most mitochondria-selective ligands are not G4-targeting. Herein, we report a new small-sized dicationic lipophilic ligand to target MMP and mitochondrial DNA G4s to enhance drug delivery for anticancer. The ligand showed marked alteration of mitochondrial gene expression and substantial induction of ROS production, mitochondrial dysfunction, DNA damage, cellular senescence, and apoptosis. The ligand also exhibited high anticancer activity against HCT116 cancer cells (IC50, 3.4 μM) and high antitumor efficacy in the HCT116 tumor xenograft mouse model (∼70% tumor weight reduction).

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“…However, in the absence of RNA, the probe binds to DNA, in turn becoming a nuclear stain. This preferential colocalization of TOR-G4 with RNA makes the molecule one of a limited number of probes well suited for studying G4 RNA within cells using fluorescence microscopy. − …”

Section: Resultsmentioning

confidence: 99%

“…Historically, quadruplex research has been focused on DNA G4s, which is reflected in the greater availability of fluorescent probes that exhibit nuclear staining . In comparison, visualization of RNA G4s (rG4s) is still a relatively new area of study. − Previously, it was suggested that RNA G4s may even be globally unfolded within cells due to the persistent activity of G4 helicases . However, more recent studies have provided extensive evidence that rG4s not only exist within cells but may be pivotal for mediating RNA interactions with proteins that control processes such as splicing, translation, and stress granule formation. − …”

Section: Discussionmentioning

confidence: 99%

Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy

Robinson,

Stenspil,

Maleckaite

et al. 2023

J. Am. Chem. Soc.

View full text Add to dashboard Cite

Over the past decade, appreciation of the roles of G-quadruplex (G4) structures in cellular regulation and maintenance has rapidly grown, making the establishment of robust methods to visualize G4s increasingly important. Fluorescent probes are commonly used for G4 detection in vitro; however, achieving sufficient selectivity to detect G4s in a dense and structurally diverse cellular environment is challenging. The use of fluorescent probes for G4 detection is further complicated by variations of probe uptake into cells, which may affect fluorescence intensity independently of G4 abundance. In this work, we report an alternative small-molecule approach to visualize G4s that does not rely on fluorescence intensity switch-on and, thus, does not require the use of molecules with exclusive G4 binding selectivity. Specifically, we have developed a novel thiazole orange derivative, TOR-G4, that exhibits a unique fluorescence lifetime when bound to G4s compared to other structures, allowing G4 binding to be sensitively distinguished from non-G4 binding, independent of the local probe concentration. Furthermore, TOR-G4 primarily colocalizes with RNA in the cytoplasm and nucleoli of cells, making it the first lifetime-based probe validated for exploring the emerging roles of RNA G4s in cellulo.

show abstract

A Cytoplasm‐Specific Fluorescent Ligand for Selective Imaging of RNA G‐Quadruplexes in Live Cancer Cells

Cited by 7 publications

References 54 publications

Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

Cellular visualization of G-quadruplex RNA via fluorescence lifetime imaging microscopy

Mitochondria-Selective Dicationic Small-Molecule Ligand Targeting G-Quadruplex Structures for Human Colorectal Cancer Therapy

Cellular Visualization of G-Quadruplex RNA via Fluorescence- Lifetime Imaging Microscopy

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