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

[Image: see text] 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 influence 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 work demonstrates that resistive pulse measurements using polymer-coated solid-state nanopores enable single-particle-level characterization of the size and shape of individual αSyn oligomers in solution within minutes. A comparison of the resulting size distribution with single-particle analysis by transmission electron microscopy and mass photometry reveals good agreement with superior resolution by nanopore-based characterization. Moreover, nanopore-based analysis has the capability to combine rapid size analysis with an approximation of the oligomer shape. Applying this shape approximation to putatively toxic oligomeric species that range in size from 18 ± 7 aggregated monomers (10S) to 29 ± 10 aggregated monomers (15S) and in concentration from picomolar to nanomolar revealed oligomer 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.