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.

Synthesis, characterization, surface properties and energy device characterstics of 2D borocarbonitrides, (BN)xC1−x, covalently cross-linked with sheets of other 2D materials

“…Chemical tagging of the carboxyl groups on the surfaces of BCN and cross-linked BCN assemblies was carried out by using 1-(bromoacetyl)pyrene following the procedure. To a 2 mg sample, 2 mL of 1.35 mM solution of 1-(bromoacetyl)pyrene in DMF, K 2 CO 3 (540 mL of 7.24 mM) and KI (450 mL of 6.02 mM) were added and stirred in dark at 50 C for 12 h. 13,14 Aer the reaction, the resulting solution was centrifuged and the supernatant was transferred to a ask for photoluminescence studies.…”

“…13, 14 The number of carboxyl groups present on the BCN surface was estimated by the calibration graph shown in Fig. 3a Fig.…”

Reversible manipulation of the G-quadruplex structures and enzymatic reactions through supramolecular host–guest interactions

“…CB7 can bind to the guests with high biocompatibility and specificity. Importantly, it has been successfully used in many biological applications, such as fishing for plasma membrane proteins (35), regulation of bioorthogonal catalysis in cells (36) and the fabrication of biomimetic systems (37). …”

Label-Free Fluorescent Determination of Sunset Yellow in Soft Drinks Based on an Indicator-Displacement Assay

“…The popularity of CB[ ] is ascribed to their novel properties in the formation of inclusion complexes with various guest molecules with high selectivity and high binding constant in aqueous solution [17][18][19][20][21]. In recent years, fluorescence enhancement approach with IDA principle based on CB[ ] host-guest complexation has been used extensively for the determination of pesticides [22,23], drugs [24], and other compounds [25].…”