High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events

Bokyoung Gong; Bong Kyu Choi; Jae Yeol Kim; Dinesh Shetty; Young Ho Ko; Narayanan Selvapalam; Nam Ki Lee; Kimoon Kim

doi:10.1021/jacs.5b05385

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events

Bokyoung Gong
, Bong Kyu Choi
, Jae Yeol Kim
, Dinesh Shetty
, Young Ho Ko
, Narayanan Selvapalam
, Nam Ki Lee
, Kimoon Kim

Chemistry

Pohang University of Science and Technology

Research output: Contribution to journal › Article › peer-review

85 Scopus citations

Abstract

Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

Original language	British English
Pages (from-to)	8908-8911
Number of pages	4
Journal	Journal of the American Chemical Society
Volume	137
Issue number	28
DOIs	https://doi.org/10.1021/jacs.5b05385
State	Published - 22 Jul 2015

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10.1021/jacs.5b05385

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title = "High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events",

abstract = "Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.",

author = "Bokyoung Gong and Choi, \{Bong Kyu\} and Kim, \{Jae Yeol\} and Dinesh Shetty and Ko, \{Young Ho\} and Narayanan Selvapalam and Lee, \{Nam Ki\} and Kimoon Kim",

note = "Publisher Copyright: {\textcopyright} 2015 American Chemical Society.",

year = "2015",

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doi = "10.1021/jacs.5b05385",

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T2 - Observation of Flickering Fusion Events

AU - Gong, Bokyoung

AU - Choi, Bong Kyu

AU - Kim, Jae Yeol

AU - Shetty, Dinesh

AU - Ko, Young Ho

AU - Selvapalam, Narayanan

AU - Lee, Nam Ki

AU - Kim, Kimoon

PY - 2015/7/22

Y1 - 2015/7/22

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AB - Fluorescence-based single-vesicle fusion assays provide a powerful method for studying mechanisms underlying complex biological processes of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated vesicle fusion and neurotransmitter release. A crucial element of these assays is the ability of the fluorescent probe(s) to reliably detect key intermediate events of fusion pore opening and content release/mixing. Here, we report a new, reliable, and efficient single-vesicle content-mixing assay using a high affinity, fluorophore tagged host-guest pair, cucurbit[7]uril-Cy3 and adamantane-Cy5 as a fluorescence resonance energy transfer (FRET) pair. The power of these probes is demonstrated by the first successful observation of flickering dynamics of the fusion pore by in vitro assay using neuronal SNARE-reconstituted vesicles.

UR - https://www.scopus.com/pages/publications/84937691923

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JO - Journal of the American Chemical Society

JF - Journal of the American Chemical Society

IS - 28

ER -

High Affinity Host-Guest FRET Pair for Single-Vesicle Content-Mixing Assay: Observation of Flickering Fusion Events

Abstract

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