Advances in <scp>G</scp>‐quadruplexes‐based fluorescent imaging

Han, Jiao-Na; Ge, Mingmin; Chen, Pengfei; Kuang, Shi; Nie, Zhou

doi:10.1002/bip.23528

Cited by 11 publications

(9 citation statements)

References 114 publications

(163 reference statements)

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“…55,56 Untangling the role of G4s in cellular processes has been facilitated by G4 visualization using either small-molecule fluorescent probes or fluorescently labeled antibodies. [12][13][14][15][16][17]39,57 Historically, quadruplex research has been focused on DNA G4s, which is reflected in the greater availability of fluorescent probes that exhibit nuclear staining. 58 In comparison, visualization of RNA G4s (rG4s) is still a relatively new area of study.…”

Section: ■ Discussionmentioning

confidence: 99%

“…The formation of G4 structures in DNA and RNA has been linked to multiple regulatory pathways across the cell, in increasingly diverse and compelling ways. − G4s are thus promising therapeutic targets for diseases, including cancer and neurodegeneration. , Untangling the role of G4s in cellular processes has been facilitated by G4 visualization using either small-molecule fluorescent probes or fluorescently labeled antibodies. − ,, …”

Section: Discussionmentioning

confidence: 99%

“…The first direct proof of G4 formation within cells was provided using G4-specific antibodies visualized by immunostaining. − In parallel, several small-molecule “switch-on” probes have been reported that become fluorescent upon binding to G4s. − While such fluorescence intensity probes are very useful for in vitro studies, their use for understanding G4 biology in cells presents several challenges. Exceptionally high G4 selectivity is required for such probes to work effectively within cells, due to the large abundance of non-G4 secondary structures that may result in a smaller “switch-on” which is, however, sufficient to produce false positive fluorescent signals. , Not only is this high G4 binding affinity difficult to achieve, but may in fact alter natural G4 formation within cells .…”

Section: Introductionmentioning

confidence: 99%

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

Robinson,

Stenspil,

Maleckaite

et al. 2023

J. Am. Chem. Soc.

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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.

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Section: ■ Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Robinson,

Stenspil,

Maleckaite

et al. 2023

J. Am. Chem. Soc.

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“…Non-B DNA secondary structures play many roles in vivo , such as Holliday junctions in recombination and slipped structures in mutagenesis. − Most recently, G-quadruplexes were proposed to play an important role in telomere maintenance and modulating transcription and translation, as well as causing genome instability. − Unfortunately, most non-B DNA structures exist transiently in vivo and are difficult to detect and locate. Small-molecule and antibody probes for detecting G-quadruplexes in vivo have been reported, − some of which drive the formation of the structures that they seek to probe and/or require fixation of the cells, which chemically alters the DNA . Many of the probes also bind to multiple G-quadruplex conformations with varying selectivity.…”

Section: Introductionmentioning

confidence: 99%

Post- and Pre-Radiolabeling Assays for anti Thymidine Cyclobutane Dimers as Intrinsic Photoprobes of Various Types of G-Quadruplexes, Reverse Hoogsteen Hairpins, and Other Non-B DNA Structures

Gutierrez-Bayona,

Scruggs,

Yang

et al. 2023

Biochemistry

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G-quadruplexes are thought to play an important role in gene regulation and telomere maintenance, but developing probes for their presence and location is challenging due to their transitory and highly dynamic nature. The majority of probes for G-quadruplexes have relied on antibody or small-molecule binding agents, many of which can also alter the dynamics and relative populations of G-quadruplexes. Recently, it was discovered that ultraviolet B (UVB) irradiation of human telomeric DNA and various G-quadruplex forming sequences found in human promoters, as well as reverse Hoogsteen hairpins, produces a unique class of non-adjacent anti cyclobutane pyrimidine dimers (CPDs). Therefore, one can envision using a pulse of UVB light to irreversibly trap these non-B DNA structures via anti CPD formation without perturbing their dynamics, after which the anti CPDs can be identified and mapped. As a first step toward this goal, we report radioactive post- and pre-labeling assays for the detection of non-adjacent CPDs and illustrate their use in detecting trans,anti T=(T) CPD formation in a human telomeric DNA sequence. Both assays make use of snake venom phosphodiesterase (SVP) to degrade the trans,anti T=(T) CPD-containing DNA to the tetranucleotide pTT=(pTT) corresponding to CPD formation between the underlined T′s of two separate dinucleotides while degrading the adjacent syn TT CPDs to the trinucleotide pGT=T. In the post-labeling assay, calf intestinal phosphodiesterase is used to dephosphorylate the tetranucleotides, which are then rephosphorylated with kinase and [32P]-ATP to produce radiolabeled mono- and diphosphorylated tetranucleotides. The tetranucleotides are confirmed to be non-adjacent CPDs by 254 nm photoreversion to the dinucleotide p*TT. In the pre-labeling assay, radiolabeled phosphates are introduced into non-adjacent CPD-forming sites by ligation prior to irradiation, thereby eliminating the dephosphorylation and rephosphorylation steps. The assays are also demonstrated to detect the stereoisomeric cis,anti T=(T) CPD.

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“…To date, a series of fluorescent molecules have been designed and developed as optical probes for the visualization of G4s. [12][13][14][15][16] However, most of them target the nucleus, and there are few probes, especially with two-photon fluorescence properties and fluorescence lifetime responses, that can specifically target cytoplasmic G4s, such as mitochondrial DNA-G4s and RNA-G4s. [17][18][19] We previously designed an organic small-molecule fluorescent probe, NBTE, which has been shown to have significantly distinguishable differences in fluorescence quantum yield (F) and fluorescence lifetime responses between DNA G4s and other DNA topologies.…”

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confidence: 99%