Simultaneous Determination of the Size and Shape of Single α-Synuclein Oligomers in Solution

Abstract

Soluble oligomers of amyloid-forming proteins are implicated as toxic species in the context of several neurodegenerative diseases. Since the size and shape of these oligomers influences their toxicity, their biophysical characterization is essential for a better understanding of the structure-toxicity relationship. Amyloid oligomers are difficult to characterize by conventional approaches due to their heterogeneity in size and shape, their dynamic aggregation process, and their low abundance. This paper demonstrates that resistive-pulse measurements using polymer-coated solid-state nanopores enable single-particle level characterization of the size and shape of individual Syn oligomers in solution within minutes. A comparison of the resulting size distribution with single-particle analysis by transmission electron microscopy and mass photometry reveals that nanopore-based characterization agrees well with both methods, while providing better size resolution and elucidating that Syn samples are composed of stable oligomer sub-populations that contain multiples of approximately 12 monomers (i.e., 12-, 24-, 48-, 60-, 84-mers). Applying the unique capability of nanopores to approximate particle size and shape to picomolar concentrations of Syn oligomers in the putatively toxic size range, revealed shapes that agree well with previous estimates by cryo-EM with the added advantage that nanopore-based analysis occurs rapidly, in solution, and has the potential to become a widely accessible technique.