While cryo-electron microscopy (cryoEM) has progressed towards imaging cells in near-native states, effective labeling of biomolecules remains a major bottleneck. The intracellular environment is highly complex, driving non-specific interactions and label-associated perturbations that hinder accurate biomacromolecule localization.
An ideal cryoEM label must provide easy visibility and selective target binding. Colloidal gold nanoparticle (AuNPs) labels are widely used due to their strong electron contrast and tunable surface chemistry, but suffer from non-specific and non-stoichiometric labeling, size dispersity, and susceptibility to ligand exchange in intracellular environments.
Atomically precise gold nanoclusters (AuNCs) have emerged as promising alternatives due to their monodispersity and well-defined ligand shells. NanogoldTM (a 1.4 nm Au55NC) is a common, commercially available label, but evidence suggests its ligand shell—likely composed of triphenylphosphine (PPh3) —is prone to exchange with intracellular thiols such as glutathione. This instability can result in aggregation, protein corona formation, and disruption of native cellular conditions, highlighting the need for more robust and specific AuNC-based labels.
This proposal aims to develop a next-generation AuNC cryoEM label that simultaneously enhances ligand exchange resistance and target specificity. The design leverages a water-soluble, para-mercaptobenzoic acid (pMBA)-protected AuNC scaffold modified with a mixed ligand shell. This ligand shell incorporates a stealth component (e.g., polysarcosine) to minimize non-specific interactions, alongside a biomolecular anchor to enable selective targeting. Ligand exchange resistance will be evaluated using a two-dimensional titration framework, probing AuNC stability under increasing glutathione concentrations and varying ionic and protein conditions representative of biological environments. Target specificity will be assessed using ribosomal labeling as a proof of concept. Functionalized AuNCs will be incubated in mixtures containing ribosomes and competing biomacromolecules, and transmission electron microscopy (TEM) will be used to quantify labeling selectivity and assess any structural or morphological changes in the AuNCs.
Overall, this work seeks to establish a design framework for stable, specific AuNC-based labels that enable more accurate and minimally perturbative intracellular cryoEM imaging.
