Biology subjects

Rocamonde Lago, I.

Publications and source records attributed to Rocamonde Lago, I..

2 recordsLinked to original sources

bioRxiv2026

Resolving multivalent antibody binding states with defined spatial antigen-patterning

Multivalent interactions are fundamental to many biological systems, including how antibodies bind to their respective antigens. Still, their accumulated binding strength, i.e. avidity, resulting from binding and rebinding events of multiple interacting points, remains difficult to measure. Classical assay platforms like ELISA and SPR lack control over antigen positioning, limiting the resolution of the binding characteristics that shape biological outcomes at nanoscale. Here, we present PANMAP, a planar and plate-based assay inspired by ELISA that uses DNA origami to present antigens at defined nanoscale patterns, enabling direct measurements of spatially resolved binding events, termed avidity profiles. Using IgG antibodies as a model system, PANMAP distinguishes between monovalent and bivalent binding states by combining equilibrium absorbance measurements with a simple biophysical model. The avidity profiles reveal how the balance shifts between monovalent and bivalent interactions as a function of antibody concentration and antigen spacing, with intermediate separation distances favoring bivalent binding events and excluded at both near and far distances. This spatial profiling allows decoupling of affinity dependency from avidity profiles to reveal how spatial constraints influence binding equilibria. Our approach fills a longstanding gap in multivalent interaction measurement and offers a new tool for antibody engineering, development of multivalent reagents, therapeutic screening, and mechanistic immunology.

immunology↗

bioRxiv2022

Notch engagement by Jag1 nanoscale clusters indicates a force-independent mode of activation

The Notch signaling pathway is a cell-cell communication system with fundamental roles in embryonic development and the nervous system. The model of Notch receptor activation that is currently most accepted, involves a force-induced conformation change at the negative regulatory region of the receptor, the subsequent recruitment of ADAM metalloproteases and a cleavage cascade that releases the Notch intracellular domain. Here, we define conditions that enable force-independent Notch activation through the formation of soluble, long-lived, multivalent ligand-receptor complexes. To investigate how ligand valency affects activation of Notch receptors, we treated iPSc-derived neuroepithelial stem-like (lt-NES) cells with different spatially defined, molecularly precise ligand nanopatterns on DNA origami nanostructures. Our data indicate that Notch signaling is activated via stimulation with multivalent clusters of the ligand Jag1, and even multivalent chimeric structures where some Jag1 proteins are replaced by other binders that do not target Notch. The findings are corroborated by systematic elimination, through experimental control, of several confounding factors that potentially could generate forces, including electrostatic interactions, endocytosis and non-specific binding. Taken together, our data suggest a model where Jag1 ligands are able to activate Notch receptors upon prolonged binding, which subsequently triggers downstream signaling in a force independent manner. These findings reveal a distinct mode of activation of Notch and could lay the foundation for the development of soluble Notch agonists that currently remain elusive.

cell biology↗