Background

PSD-95 is a key scaffolding protein and a widely used postsynaptic marker for excitatory synapses. Within the postsynaptic density (PSD), PSD-95 assembles into dense nanodomains that recruit both AMPA and NMDA receptors in alignment with the presynaptic active zone, thereby facilitating and regulating efficient synaptic transmission. Dysregulation of PSD-95 has been associated with cognitive and learning deficits linked to neurodevelopmental disorders such as schizophrenia and autism [3].

The highly crowded environment of the PSD presents a major challenge for reliable antibody-based labeling, as the large size of full-length antibodies introduces significant steric hindrance and limits access to epitopes deep within the nanodomain. This often results in biased labeling toward peripheral molecules. Additionally, full-length antibodies introduce a linkage error of up to ~12 nm, and commonly used primary–secondary antibody complexes can extend this to ~24 nm. While negligible in diffraction-limited microscopy, such displacement becomes substantial in super-resolution techniques with sub-50 nm resolution.

To overcome these limitations, we developed Sylites using a peptide microarray approach [4]. Sylites are synthetic bidentate peptide probes with nanomolar affinity for key postsynaptic density markers: PSD-95 at excitatory synapses and Gephyrin at inhibitory synapses. Unlike conventional full-length antibodies (~150 kDa) or nanobodies (~15 kDa) [5], the ~3 kDa Sylite peptides minimize dye offset, significantly reducing linkage error and enabling precise, spot-on labeling for high-resolution microscopy. Their small size also greatly enhances tissue penetration and labeling efficiency, particularly within densely packed synaptic environments.

eSylites were designed by mimicking and dimerizing the C-terminal tail of GluN2B, a subunit of NMDA receptors. This confers nanomolar affinity and high specificity for the PDZ1 and PDZ2 domains of PSD-95 through avidity. We demonstrate that eSylites offer superior localization accuracy and structural resolution of postsynaptic nanodomains compared to conventional antibodies.

In parallel, iSylites were developed as dimers derived from the GlyR β subunit binding sequence, targeting the C-terminal E-domain of Gephyrin, which forms a dimeric structure. The modifications, together with avidity-based design, enhance binding affinity to Gephyrin up to 46,000-fold [6], enabling reliable and specific labeling of inhibitory postsynaptic densities. Together with compact synthetic probes developed by the Hamachi lab for GABA_ARs and GluRs [7], Sylites offer a complete toolbox for the offset-free labeling of inhibitory and excitatory neurotransmitter receptors, together with their underlying post-synaptic scaffolds.

Both eSylites and iSylites take advantage of the fractional occupancy of scaffolding protein binding sites [8,9]. While this functional selectivity is negligible in diffraction-limited imaging, it can reveal additional insights into the reserve pool of scaffold proteins when super-resolution techniques with resolution beyond 10 nm are applied. Currently, Sylites face limitations in direct stochastic optical reconstruction microscopy (dSTORM) imaging due to dense labeling, which may induce energy transfer mechanisms that compromise localization precision and photostability in dSTORM buffers [10]. Nevertheless, their fully synthetic origin and modular design make Sylites highly adaptable for specific experimental applications. In the future, advanced imaging techniques such as expansion microscopy (ExM) combined with DNA-PAINT or optimized dSTORM conditions may further enhance the utility of Sylites in nanoscale synaptic mapping.