2h3 x sc dodd  (Developmental Studies Hybridoma Bank)

Journal: bioRxiv

Article Title: Distinct allosteric remodeling of HIV-1 Env dynamics on virions by gp41-directed antibodies reveals two modes of neutralization

doi: 10.64898/2026.01.27.702099

Figure Lengend Snippet: ( A ) Genetic code expansion via amber suppression. Amber suppressor tRNA and tRNA synthetase (tRNA Pyl /NESPylRS AF ) incorporate the ncAA trans-cyclooct-2-en-L-lysine (TCO*A) at TAG codons engineered into Env (S401 TAG in gp120 and R542 TAG in gp41) on intact virions produced in mammalian cells. The supply of TCO*A to transfected cells enables its incorporation at the designated positions, providing reactive handles for subsequent fluorophore conjugation. ( B ) Bioorthogonal click labeling via SPIEDAC. Tetrazine-conjugated Cy3 and Cy5 derivatives (LD555-TTZ and LD655-TTZ) react with the strained alkene of TCO*A through strain-promoted inverse electron-demand Diels–Alder cycloaddition (SPIEDAC). The conjugated fluorophores (dyes) are depicted as red spheres. TCO*A Functional groups are shown in the modeled membrane-present Env trimers (right panel).

Article Snippet: The ncAA trans-cyclooct-2-en-L-lysine (TCO*A; SiChem #SC8008) was added to the culture medium at a final concentration of 250 μM.

Techniques: Produced, Transfection, Conjugation Assay, Labeling, Functional Assay, Membrane