Bokyoung Gong†‡, Bong-Kyu Choi §, Jae-Yeol Kim ∥, Dinesh Shetty†, Young Ho Ko†, Narayanan Selvapalam†, Nam Ki Lee*§∥, and Kimoon Kim*†‡#
†Center for Self-Assembly and Complexity (CSC), Institute for Basic Science (IBS), ‡Department of Chemistry, §School of Interdisciplinary Bioscience and Bioengineering, and ∥Department of Physics, #Division of Advanced Materials Science, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea
* Correspondence to Nam Ki Lee, Kimoon Kim
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.