Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Summers, Peter A.; Lewis, Benjamin F.; González‐García, Jorge; Porreca, Rosa María; Lim, Aaron H. M.; Cadinu, Paolo; Martín-Pintado, Nerea; Mann, David J.; Edel, Joshua B.; Vannier, Jean Baptiste; Kuimova, Marina K.; Vilar, Ramón

doi:10.1038/s41467-020-20414-7

Cited by 125 publications

(99 citation statements)

References 70 publications

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“…Interestingly, a recently reported magnetic tweezers study of the promoter region of the c-kit oncogene demonstrated that negative superhelical strain can also drive the B-form to GQ structural transition 53 . The results of our mechanical analysis of telomeric D-loops suggest the process of strand invasion at telomere DNA targets may provide an opportunity for GQ structures to fold in vivo, as has recently been reported by live cell imaging 54,55 .…”

Section: Discussionsupporting

confidence: 72%

Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

Long

Shastry

et al. 2021

Preprint

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Telomeres are essential chromosome end capping structures that safeguard the genome from dangerous DNA processing events. DNA strand invasion occurs during vital transactions at telomeres, including telomere length maintenance by the alternative lengthening of telomeres (ALT) pathway. During telomeric strand invasion, a single stranded guanine-rich (G-rich) DNA invades at a complimentary duplex telomere repeat sequence forming a displacement loop (D-loop) in which the displaced DNA consists of the same G-rich sequence as the invading single stranded DNA. Single stranded G-rich telomeric DNA readily folds into stable, compact, structures called G-quadruplexes (GQ) in vitro, and is anticipated to form within the context of a D-loop; however, evidence supporting this hypothesis is lacking. Here we report a magnetic tweezers assay that permits the controlled formation of telomeric D-loops (TDLs) within uninterrupted duplex human telomere DNA molecules of physiologically relevant lengths. Our results are consistent with a model wherein the displaced single stranded DNA of a TDL folds into a GQ. This study provides new insight into telomere structure and establishes a framework for development of novel therapeutics designed to target GQs at telomeres in cancer cells.

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Section: Discussionsupporting

confidence: 72%

Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

Long

Shastry

et al. 2021

Preprint

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“…A large number of metallosalens do not present fluorescent properties suitable for microscopy imaging studies with live cells. To circumvent this limitation, Vilar and coworkers developed recently a new quantitative fluorescence lifetime-based displacement assay to visualize the strength and the rates of the interaction of these small molecules with G4 in live cells [91]. This innovative assay relies on the use of the molecule DAOTA-M2 (Figure 10) that shows remarkably different fluorescence lifetime when bound to G4 structures, as compared to duplex or single stranded DNA.…”

Section: Metal-based G4 Binders For Cancer Theranostics: Fluorescent Probes and Anticancer Agents 21 Complexes With Schiff Basesmentioning

confidence: 99%

Metal-Based G-Quadruplex Binders for Cancer Theranostics

et al. 2021

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The ability of fluorescent small molecules, such as metal complexes, to selectively recognize G-quadruplex (G4) structures has opened a route to develop new probes for the visualization of these DNA structures in cells. The main goal of this review is to update the most recent research efforts towards the development of novel cancer theranostic agents using this type of metal-based probes that specifically recognize G4 structures. This encompassed a comprehensive overview of the most significant progress in the field, namely based on complexes with Cu, Pt, and Ru that are among the most studied metals to obtain this class of molecules. It is also discussed the potential interest of obtaining G4-binders with medical radiometals (e.g., 99mTc, 111In, 64Cu, 195mPt) suitable for diagnostic and/or therapeutic applications within nuclear medicine modalities, in order to enable their theranostic potential.

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“…To address the challenge, some G-rich sequences are exploited in constructing label-free fluorescence biosensors, due to the G-rich sequence being able to self-assemble into a G-quadruplex structure. As we known, the G-quadruplex is a non-canonical DNA structure and noncovalently combines with small molecule ligands (such as thioflavin T or hemin) to form label-free probes, which have been widely used in biological progress monitoring and biomolecules analysis [ 13 , 14 , 15 ], wherein their applications occur with the concomitant conformational change of the G-quadruplex. However, the control and initiation of G-quadruplex structures are often faced with some difficulties.…”

Section: Introductionmentioning

confidence: 99%

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection

Xue

Zhou

2021

Molecules

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Protein detection plays an important role in biological and biomedical sciences. The immunoassay based on fluorescence labeling has good specificity but a high labeling cost. Herein, on the basis of G-triplex molecular beacon (G3MB) and thioflavin T (ThT), we developed a simple and label-free biosensor for protein detection. The biotin and streptavidin were used as model enzymes. In the presence of target streptavidin (SA), the streptavidin hybridized with G3MB-b (biotin-linked-G-triplex molecular beacon) perfectly and formed larger steric hindrance, which hindered the hydrolysis of probes by exonuclease III (Exo III). In the absence of target streptavidin, the exonuclease III successively cleaved the stem of G3MB-b and released the G-rich sequences which self-assembled into a G-triplex and subsequently activated the fluorescence signal of thioflavin T. Compared with the traditional G-quadruplex molecular beacon (G4MB), the G3MB only needed a lower dosage of exonuclease III and a shorter reaction time to reach the optimal detection performance, because the concise sequence of G-triplex was good for the molecular beacon design. Moreover, fluorescence experiment results exhibited that the G3MB-b had good sensitivity and specificity for streptavidin detection. The developed label-free biosensor provides a valuable and general platform for protein detection.

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Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Cited by 125 publications

References 70 publications

Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

Single-molecule Mechanical Analysis of Strand Invasion in Human Telomere DNA

Metal-Based G-Quadruplex Binders for Cancer Theranostics

Label-Free Fluorescence Molecular Beacon Probes Based on G-Triplex DNA and Thioflavin T for Protein Detection

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