The effects of fluorescent labels on Aβ₄₂ aggregation detected by fluorescence correlation spectroscopy

Abstract

Fluorescence‐based methods are promising for measuring amyloid beta (Aβ) oligomers, given their capacity to analyse a sample at the single‐molecule level. As the attachment of fluorescent labels may influence the biochemical properties of the Aβ oligomers, the effects of fluorescent labels on Aβ oligomers must be evaluated. In this paper, we compared the impacts of five different fluorescent dyes on the aggregation of Aβ42 oligomers using fluorescence correlation spectroscopy (FCS). We found that fluorescent labels of BODIPY® FL‐C5 (BP), N‐hydroxysuccinimide rhodamine B ester (RB) and rhodamine B isothiocyanate (RITC) increased the propensity of labelled Aβ42 oligomers to aggregate, whereas 6‐(fluorescein‐5‐carboxamido) hexanoic acid succinimidyl ester (5‐SFX) and fluorescein 5(6)‐isothiocyanate (5(6)‐FITC) decreased the propensity of labelled Aβ42 oligomers to aggregate. This difference originated from the different electric charges and hydrophobicity of the fluorescent dyes. These results provide valuable information for establishing different aggregation models for Aβ42 oligomers in vitro using FCS.

Clusterin inhibits Aβ42 aggregation through a “strawberry model” as detected by FRET‐FCS

“…Before each experiment, after DMSO was added to one aliquot of fluorescently labeled Aβ 42 monomer, the DMSO solutions of one aggregation model (such as Aβ 42/RB and Aβ 42/Atto647 ) were mixed together to a final concentration of 100 μM. After ultrasonication for 2 min, the mixed stock solution was diluted in PBS buffer 1–100 to produce oligomers (The produced oligomers are prefibrillar oligomers, which is consistent with the literature and our previous work (Kayed et al., 2003; Ly et al., 2013; Zheng et al., 2018; Zheng et al., 2016) at a concentration of 1 μM. Finally, the FRET‐FCS measurement was performed at a concentration of 1 nM Aβ 42 to obtain a high signal‐to‐noise ratio.…”

“…Fluorescently labeled Aβ 42 was prepared according to the previously described method (Zheng et al, 2016(Zheng et al, , 2018. Synthetic Aβ 42 peptide (0.5 mg, purity ≥98%; Sangon, Shanghai, China) was dissolved in hexafluoro-2-propanol (HFIP, Cat#105228, Sigma, St. Louis, USA) to a final concentration of 1 mg/ml and incubated at room temperature with gentle shaking for 24 hr.…”

“…So, it should be demonstrated clearly what is the molecular mechanism of the interaction of clusterin and Aβ 42 . In recent years, our research interest has focused on the aggregation of Aβ 42 at the single‐molecule level based on fluorescence detection (Zheng et al., 2016, 2018), and here, we tried to investigate the effects of clusterin on the aggregation of Aβ 42 using florescence resonance energy transfer‐fluorescence correlation spectroscopy (FRET‐FCS).…”

“…The study of the aggregation behavior of Aβ 42 at the singlemolecule level is our research interest. In previous work (Zheng et al, 2018)…”

Single-molecule studies of amyloid proteins: from biophysical properties to diagnostic perspectives

“…TIRF has been applied for the direct visualization of amyloid aggregates. As using covalently labeling proteins might affect the aggregation process (Zheng et al, 2018) and it is challenging to tag samples extracted from in vivo sources, Horrocks et al (2016) relied on the addition of a dye (Thioflavin-T (ThT)) that non-covalently binds to oligomers and fibrils with β-sheet structures. Small β-sheet aggregates of α-syn, Aβ and tau in vitro are observed with the sizes <250 nm at the beginning of aggregation and the enriched oligomers contain on average 25 monomers.…”