Dataset Information

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

ABSTRACT: DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp; ; Stuurman; ; Vale Nature Methods, 2020). FluoroCubes, that is, compact ∼4 × 4 × 5.4 nm³ four-helix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about the FluoroCubes. First, what is the mechanism by which photostability is enhanced? Second, are FluoroCubes compatible with Förster resonance energy transfer (FRET) and similar techniques? We use single particle photobleaching studies to show that photostability arises through interactions between the fluorophores and the four-helix DNA bundle. Supporting this, we discover that smaller ∼4 × 4 × 2.7 nm³ FluoroCubes also confer ultraphotostability. However, we find that certain dye-dye interactions negatively impact FluoroCube performance. Accordingly, 4-dye FluoroCubes lacking these interactions perform better than 6-dye FluoroCubes. We also demonstrate that FluoroCubes are compatible with FRET and dark quenching applications.

SUBMITTER: Blanchard AT

PROVIDER: S-EPMC10080265 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

Blanchard Aaron T AT Li Zi Z Duran Elizabeth C EC Scull Catherine E CE Hoff J Damon JD Wright Keenan R KR Pan Victor V Walter Nils G NG

Nano letters 20220726 15

DNA-based FluoroCubes were recently developed as a solution to photobleaching, a ubiquitous limitation of fluorescence microscopy (Niekamp; ; Stuurman; ; Vale <i>Nature Methods</i>, 2020). FluoroCubes, that is, compact ∼4 × 4 × 5.4 nm<sup>3</sup> four-helix bundles coupled to ≤6 fluorescent dyes, remain fluorescent up to ∼50× longer than single dyes and emit up to ∼40× as many photons. The current work answers two important questions about the FluoroCubes. First, what is the mechanism by which p ...[more]

PMID: 35881934

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Project description:Dark quenchers are chromophores that primarily relax from the excited state to the ground state nonradiatively (i.e., are dark). As a result, they can serve as acceptors for Förster resonance energy transfer experiments without contributing significantly to background in the donor-emission channel, even at high concentrations. Although the advantages of dark quenchers have been exploited for ensemble bioassays, no systematic single-molecule study of dark quenchers has been performed, and little is known about their photophysical properties. Here, we present the first systematic single-molecule study of dark quenchers in conjunction with fluorophores and demonstrate the use of dark quenchers for monitoring multiple interactions and distances in multichromophore systems. Specifically, using double-stranded DNA standards labeled with two fluorophores and a dark quencher (either QSY7 or QSY21), we show that the proximity of a fluorophore and dark quencher can be monitored using the stoichiometry ratio available from alternating laser excitation spectroscopy experiments, either for single molecules diffusing in solution (using a confocal fluorescence) or immobilized on surfaces (using total-internal-reflection fluorescence). The latter experiments allowed characterization of the dark-quencher photophysical properties at the single-molecule level. We also use dark-quenchers to study the affinity and kinetics of binding of DNA Polymerase I (Klenow fragment) to DNA. The measured properties are in excellent agreement with the results of ensemble assays, validating the use of dark quenchers. Because dark-quencher-labeled biomolecules can be used in total-internal-reflection fluorescence experiments at concentrations of 1 μM or more without introducing a significant background, the use of dark quenchers should permit single-molecule Förster resonance energy transfer measurements for the large number of biomolecules that participate in interactions of moderate-to-low affinity.

Dataset Information

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

Publications

Ultra-photostable DNA FluoroCubes: Mechanism of Photostability and Compatibility with FRET and Dark Quenching.

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