Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Wednesday Speaker Abstracts

SINGLE-MOLECULE INSIGHTS INTO THE DYNAMIC HUMAN RNA INTERFERENCE MACHINERY USING SLAM-FRET

Dina Grohman 1 ; 1 University of Regensburg, Regensburg-Galgenberg, Germany

Human cellular processes are driven by molecular recognition, conformational changes, and dynamic interactions. Because proteins exist in multiple chemical states and are extensively regulated by posttranslational modifications such as phosphorylation, understanding their function requires studying them in their native form. While single-molecule FRET (smFRET) is a powerful tool for probing biomolecular structure and dynamics, its application to the human proteome has been limited by the lack of methods for site-specific fluorescent labeling of native, non-recombinant mammalian proteins. Here, I present SLAM-FRET, a bioorthogonal chemistry based method for site-specific fluorescent labeling of native human proteins. I will show how this advance enables single-molecule studies of key regulatory pathways in human cells, focusing on RNA silencing (RNA interference, RNAi). The RNA silencing patwhay regulates the expression of most human genes and has become a widely used research tool and even is used as therapeutic modality. Yet, several fundamental mechanistic aspects of the pathway have remained unresolved. At its core is human Argonaute 2 (Ago2), a protein that has long been inaccessible to single-molecule interrogation. Using SLAM-FRET, we made native Ago2 amenable to smFRET analysis, revealing its structural states and conformational dynamics. These studies uncovered fundamental mechanisms of RNAi, including how Ago2 interacts with RNA substrates, how Ago2 engages with Dicer during small RNA loading, and how disease associated Ago2 mutations causing LESKRES syndrome alter its function.

28