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Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes

Research output: Contribution to journalArticlepeer-review

DOI

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Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes. / Quinn, Steven David; Dalgarno, Paul Allan; Cameron, Ryan T; Hedley, Gordon James; Hacker, Christian; Lucocq, John Milton; Samuel, Ifor David William; Penedo, Carlos.

In: Molecular BioSystems, Vol. 10, No. 1, 01.2014, p. 34-44.

Research output: Contribution to journalArticlepeer-review

Harvard

Quinn, SD, Dalgarno, PA, Cameron, RT, Hedley, GJ, Hacker, C, Lucocq, JM, Samuel, IDW & Penedo, C 2014, 'Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes', Molecular BioSystems, vol. 10, no. 1, pp. 34-44. https://doi.org/10.1039/C3MB70272C

APA

Quinn, S. D., Dalgarno, P. A., Cameron, R. T., Hedley, G. J., Hacker, C., Lucocq, J. M., Samuel, I. D. W., & Penedo, C. (2014). Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes. Molecular BioSystems, 10(1), 34-44. https://doi.org/10.1039/C3MB70272C

Vancouver

Quinn SD, Dalgarno PA, Cameron RT, Hedley GJ, Hacker C, Lucocq JM et al. Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes. Molecular BioSystems. 2014 Jan;10(1):34-44. https://doi.org/10.1039/C3MB70272C

Author

Quinn, Steven David ; Dalgarno, Paul Allan ; Cameron, Ryan T ; Hedley, Gordon James ; Hacker, Christian ; Lucocq, John Milton ; Samuel, Ifor David William ; Penedo, Carlos. / Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes. In: Molecular BioSystems. 2014 ; Vol. 10, No. 1. pp. 34-44.

Bibtex - Download

@article{8047637a596d454a9049e7c375dcd721,
title = "Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes",
abstract = "The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting β-amyloid aggregates (Aβ) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aβ peptides and demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of β-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aβ1-42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in F{\"o}rster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.",
keywords = "Amyloid beta-Peptides, Binding Sites, Fluorescence, Fluorescent Dyes, Humans, Kinetics, Peptide Fragments, Protein Binding, Thiazoles",
author = "Quinn, {Steven David} and Dalgarno, {Paul Allan} and Cameron, {Ryan T} and Hedley, {Gordon James} and Christian Hacker and Lucocq, {John Milton} and Samuel, {Ifor David William} and Carlos Penedo",
year = "2014",
month = jan,
doi = "10.1039/C3MB70272C",
language = "English",
volume = "10",
pages = "34--44",
journal = "Molecular BioSystems",
issn = "1742-206X",
publisher = "Royal Society of Chemistry",
number = "1",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Real-time probing of β-amyloid self-assembly and inhibition using fluorescence self-quenching between neighbouring dyes

AU - Quinn, Steven David

AU - Dalgarno, Paul Allan

AU - Cameron, Ryan T

AU - Hedley, Gordon James

AU - Hacker, Christian

AU - Lucocq, John Milton

AU - Samuel, Ifor David William

AU - Penedo, Carlos

PY - 2014/1

Y1 - 2014/1

N2 - The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting β-amyloid aggregates (Aβ) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aβ peptides and demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of β-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aβ1-42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in Förster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.

AB - The fluorescence response of the Thioflavin-T (ThT) dye and derivatives has become the standard tool for detecting β-amyloid aggregates (Aβ) in solution. However, it is accepted that ThT-based methods suffer from important drawbacks. Some of these are due to the cationic structure of ThT, which limits its application at slightly acidic conditions; whereas some limitations are related to the general use of an extrinsic-dye sensing strategy and its intrinsic requirement for the formation of a sensor-binding site during the aggregation process. Here, we introduce fluorescence-self-quenching (FSQ) between N-terminally tagged peptides as a strategy to overcome some of these limitations. Using a combination of steady-state, picosecond time-resolved fluorescence and transmission electron microscopy, we characterize the fluorescence response of HiLyte fluor 555-labelled Aβ peptides and demonstrate that Aβ self-assembly organizes the covalently attached probes in close proximity to trigger the self-quenching sensing process over a broad range of conditions. Importantly, we prove that N-terminal tagging of β-amyloid peptides does not alter the self-assembly kinetics or the resulting aggregated structures. We also tested the ability of FSQ-based methods to monitor the inhibition of Aβ1-42 aggregation using the small heat-shock protein Hsp20 as a model system. Overall, FSQ-based strategies for amyloid-sensing fill the gap between current morphology-specific protocols using extrinsic dyes, and highly-specialized single-molecule techniques that are difficult to implement in high-throughput analytical determinations. When performed in Förster resonance energy transfer (FRET) format, the method becomes a ratiometric platform to gain insights into amyloid structure and for standardizing in vitro studies of amyloid aggregation.

KW - Amyloid beta-Peptides

KW - Binding Sites

KW - Fluorescence

KW - Fluorescent Dyes

KW - Humans

KW - Kinetics

KW - Peptide Fragments

KW - Protein Binding

KW - Thiazoles

U2 - 10.1039/C3MB70272C

DO - 10.1039/C3MB70272C

M3 - Article

C2 - 24170094

VL - 10

SP - 34

EP - 44

JO - Molecular BioSystems

JF - Molecular BioSystems

SN - 1742-206X

IS - 1

ER -

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