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.
authors.affiliation: †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.
authors.forename: Bokyoung
authors.initials: B
authors.lastname: Gong
authors.affiliation: †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.
authors.forename: Bong-Kyu
authors.initials: BK
authors.lastname: Choi
authors.affiliation: †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.
authors.forename: Jae-Yeol
authors.initials: JY
authors.lastname: Kim
authors.affiliation: †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.
authors.forename: Dinesh
authors.initials: D
authors.lastname: Shetty
authors.affiliation: †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.
authors.forename: Young Ho
authors.initials: YH
authors.lastname: Ko
authors.affiliation: †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.
authors.forename: Narayanan
authors.initials: N
authors.lastname: Selvapalam
authors.affiliation: †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.
authors.forename: Nam Ki
authors.initials: NK
authors.lastname: Lee
authors.affiliation: †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.
authors.forename: Kimoon
authors.initials: K
authors.lastname: Kim
mesh_terms: D000218:Adamantane / Q000032:analysis / Q000378:metabolism; D000818:Animals; D001952:Bridged-Ring Compounds / Q000032:analysis / Q000378:metabolism; D002232:Carbocyanines / Q000032:analysis / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000032:analysis / Q000378:metabolism; D008561:Membrane Fusion; D009474:Neurons / Q000378:metabolism; D050600:SNARE Proteins / Q000032:analysis / Q000378:metabolism*; D047028:Macrocyclic Compounds; D000099246:Cucurbit(n)urils
chemical_list: D000218:Adamantane; D001952:Bridged-Ring Compounds; D002232:Carbocyanines; D005456:Fluorescent Dyes; D007093:Imidazoles; D050600:SNARE Proteins; C456276:cucurbit(7)uril; D047028:Macrocyclic Compounds; D000099246:Cucurbit(n)urils; C089187:cyanine dye 3; C085321:cyanine dye 5