Co-localization single-molecule spectroscopy reveals specificity and function of Argonaute protein-mediated pathways

Single-molecule binding assays allow the interrogation of individual macromolecules from a biological process using purified components or cellular extracts. In contrast to ensemble measurements, single-molecule assays can report the order and kinetics of individual molecular interactions. The introduction of commercial microscope assemblies designed for single-molecule imaging spurred wide adoption of this technology. However, the absence of easy-to-use software for extracting kinetic data from an image series makes analysis slow and tedious. We constructed an automated pipeline for rapid processing of co-localization single-molecule spectroscopy (CoSMoS) images while quantitatively assessing experimental data quality. All steps are controlled via a user-friendly interface; no knowledge of scripting is required. We benchmarked the pipeline by measuring the binding properties of the well-studied, DNA-guided, DNA endonuclease, TtAgo, an Argonaute protein from the Eubacterium Thermus thermophilus. We used the pipeline to extend our understanding of TtAgo by measuring the protein’s binding kinetics at different temperatures and for target DNAs containing multiple, adjacent binding sites. Using CoSMoS, we have also begun to define specificity and efficiency of mammalian Argonaute proteins loaded with microRNAs (miRNAs) and small interfering RNAs (siRNAs). Our quantitative measurements should allow prediction of complex and dynamic miRNA-mRNA regulatory networks and may facilitate the development of siRNA, miRNA, and antagomir therapeutics with high potency and target specificity.

Karina Jouravleva est invitée par Yasmina Saoudi.