METHODS article
Front. Mol. Biosci.
Sec. RNA Networks and Biology
FluoTag-EMSA: A Fast and Accessible Quantitative Method to Assess RNA-Binding Specificity Using 3′-Tagged Hybrid Duplexes
Provisionally accepted
- Ecole polytechnique federale de Lausanne, Lausanne, Switzerland
Select one of your emails
You have multiple emails registered with Frontiers:
Notify me on publication
Please enter your email address:
If you already have an account, please login
You don't have a Frontiers account ? You can register here
Electrophoretic mobility shift assay (EMSA) is widely used to study RNA-protein interactions but remains limited by safety, cost, and time constraints associated with radioactive or covalent fluorescent labeling. Here, a novel method termed FluoTag-EMSA overcomes these hurdles by providing the RNA 3'ends with short sequence tags that can be hybridized to specific complementary fluorescent DNA probes, eliminating the need for chemical or enzymatic labeling steps. Specifically, two independent sequence tags are described that do not disrupt RNA folding and allow efficient annealing to complementary oligonucleotides carrying far-red/near-infrared dyes (700 nm and 800 nm), enabling direct in-gel fluorescence detection. Both tag/probe duplexes exhibit identical thermodynamic properties and can be used interchangeably. FluoTag-EMSA is a streamlined and reproducible non-radioactive alternative method for studying RNA-protein interactions without the need for specialized equipment.
Keywords: RNA-protein interactions, RNA-Binding Protein, Electrophoretic mobility shift assay (EMSA), Non-radioactive detection, Fluorescent RNA labeling, RNA tagging, FluoTag-EMSA, Ribonucleoprotein complexes (RNPs)
Received: 17 Oct 2025; Accepted: 03 Nov 2025.
Copyright: © 2025 ROTHE and Constam. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Benjamin ROTHE, benjamin.rothe@epfl.ch
Daniel Beat Constam, daniel.constam@epfl.ch
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.