Multicolor single-molecule FRET for DNA and RNA processes

Feng, Xinyu A.; Poyton, Matthew F.; Ha, Taekjip

doi:10.1016/j.sbi.2021.03.005

Cited by 35 publications

(15 citation statements)

References 53 publications

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“…Förster resonance energy transfer (FRET) microscopy is a feasible approach to measure WWOX function in a real-time manner. FRET can be utilized to determine spatial proximity among proteins either at a single or multiple protein levels in cultured cells in a real-time mode [ 18 , 43 , 44 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 ]. For example, binding of a CFP (cyan fluorescence protein)-tagged bait (or donor) protein with a YFP (yellow fluorescence protein)-tagged target (or acceptor) protein results in energy flow from the donor to the acceptor.…”

Section: Wwox Functional Measurement By Time-lapse Fret Microscopymentioning

confidence: 99%

WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners

Liu

Nagarajan

Chiang

et al. 2022

Cells

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Tumor suppressor WWOX inhibits cancer growth and retards Alzheimer’s disease (AD) progression. Supporting evidence shows that the more strongly WWOX binds intracellular protein partners, the weaker is cancer cell growth in vivo. Whether this correlates with retardation of AD progression is unknown. Two functional forms of WWOX exhibit opposite functions. pY33-WWOX is proapoptotic and anticancer, and is essential for maintaining normal physiology. In contrast, pS14-WWOX is accumulated in the lesions of cancers and AD brains, and suppression of WWOX phosphorylation at S14 by a short peptide Zfra abolishes cancer growth and retardation of AD progression. In parallel, synthetic Zfra4-10 or WWOX7-21 peptide strengthens the binding of endogenous WWOX with intracellular protein partners leading to cancer suppression. Indeed, Zfra4-10 is potent in restoring memory loss in triple transgenic mice for AD (3xTg) by blocking the aggregation of amyloid beta 42 (Aβ42), enhancing degradation of aggregated proteins, and inhibiting activation of inflammatory NF-κB. In light of the findings, Zfra4-10-mediated suppression of cancer and AD is due, in part, to an enhanced binding of endogenous WWOX and its binding partners. In this perspective review article, we detail the molecular action of WWOX in the HYAL-2/WWOX/SMAD4 signaling for biological effects, and discuss WWOX phosphorylation forms in interacting with binding partners, leading to suppression of cancer growth and retardation of AD progression.

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Section: Wwox Functional Measurement By Time-lapse Fret Microscopymentioning

confidence: 99%

WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners

Liu

Nagarajan

Chiang

et al. 2022

Cells

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“…smFRET monitors the distance, usually between 1 and 10 nm, between single pairs of donor and acceptor fluorophores, by measuring their intensities and the extent of non-radiative energy transfer ( Ha et al, 1996 ). Unencumbered by diffraction limited resolution (∼250 nm) in typical fluorescence based single-molecule imaging experiments, smFRET has been widely employed in biophysical studies on topics ranging from replication, transcription and repair to RNA and protein conformational dynamics ( Feng et al, 2021 ). In vitro smFRET experiments usually requires immobilization of fluorescently labeled macromolecules, either DNA or protein, on the passivated flow cell surface, where excitation of fluorophores is achieved through total internal reflection ( Figure 1F ).…”

Section: Overview Of Single-molecule Techniquesmentioning

confidence: 99%

Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

Kong

Greene²

2021

Front. Cell Dev. Biol.

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DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes.

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“…In this study, we focused only on the analysis of fluorescence intensity and FRET efficiency data. The addition of complementary information from simulations or experiments (e.g., static molecular structures and other observables, such as fluorescence lifetimes, anisotropy, and more) may help to elucidate complicated or otherwise underdetermined systems 28,47,48 . Legend with all analysis tools.…”

Section: Full Complexity Of a Black-box Experimentsmentioning

confidence: 99%

Inferring kinetic rate constants from single-molecule FRET trajectories – a blind benchmark of kinetic analysis tools

Götz

Barth

Bohr

et al. 2021

Preprint

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Single-molecule FRET (smFRET) is a versatile technique to study the dynamics and function of biomolecules since it makes nanoscale movements detectable as fluorescence signals. The powerful ability to infer quantitative kinetic information from smFRET data is, however, complicated by experimental limitations. Diverse analysis tools have been developed to overcome these hurdles but a systematic comparison is lacking. Here, we report the results of a blind benchmark study assessing eleven analysis tools used to infer kinetic rate constants from smFRET trajectories. We tested them against simulated and experimental data containing the most prominent difficulties encountered in analyzing smFRET experiments: different noise levels, varied model complexity, non-equilibrium dynamics, and kinetic heterogeneity. Our results highlight the current strengths and limitations in inferring kinetic information from smFRET trajectories. In addition, we formulate concrete recommendations and identify key targets for future developments, aimed to advance our understanding of biomolecular dynamics through quantitative experiment-derived models.

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Multicolor single-molecule FRET for DNA and RNA processes

Cited by 35 publications

References 53 publications

WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners

WWOX Controls Cell Survival, Immune Response and Disease Progression by pY33 to pS14 Transition to Alternate Signaling Partners

Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

Inferring kinetic rate constants from single-molecule FRET trajectories – a blind benchmark of kinetic analysis tools

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