Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing

Joel Vega-Badillo; Phillip D Zamore; Karina Jouravleva

doi:10.1016/j.xpro.2023.102336

Protocol to measure protein-RNA binding using double filter-binding assays followed by phosphorimaging or high-throughput sequencing

STAR Protoc. 2023 Jun 3;4(2):102336. doi: 10.1016/j.xpro.2023.102336. Online ahead of print.

Authors

Joel Vega-Badillo¹, Phillip D Zamore², Karina Jouravleva³

Affiliations

¹ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address: joel.vegabadillo@umassmed.edu.
² RNA Therapeutics Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA; Howard Hughes Medical Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address: phillip.zamore@umassmed.edu.
³ RNA Therapeutics Institute, University of Massachusetts Chan Medical School, 368 Plantation Street, Worcester, MA 01605, USA. Electronic address: karina.jouravleva@umassmed.edu.

Abstract

Binding affinity quantitatively describes the strength of a molecular interaction and is reported by the equilibrium dissociation constant (K_D). Here, we present a protocol to measure K_D of mammalian microRNA-loaded Argonaute2 protein by double filter binding. We describe steps for radiolabeling target RNA, measuring concentration of binding-competent protein, setting up binding reactions, separating protein-bound RNA from protein-unbound RNA, preparing library for Illumina sequencing, and performing data analysis. Our protocol is easily applied to other RNA- or DNA-binding proteins. For complete details on the use and execution of this protocol, please refer to Jouravleva et al.¹.

Keywords: Molecular Biology; Protein Biochemistry.

Grants and funding

R35 GM136275/GM/NIGMS NIH HHS/United States