Development of bis-ANS-based modified fluorescence titration assay for IFIT/RNA studies

• Published in: Biochemical and biophysical research communications Vol. 533; no. 3; pp. 391 - 396
• Main Authors: Dobieżyńska, Anna, Miszkiewicz, Joanna, Darżynkiewicz, Zbigniew M., Tyras, Michał, Stankiewicz-Drogoń, Anna, Trylska, Joanna, Darżynkiewicz, Edward, Grzela, Renata
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.12.2020
• Subjects: Adaptor Proteins, Signal Transducing - chemistry; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Anilino Naphthalenesulfonates - chemistry; Binding Sites; Binding, Competitive; Biological Assay; Fluorescence probe; Fluorescent Dyes - chemistry; Humans; Hydrogen Bonding; IFIT; Kinetics; Molecular Docking Simulation; Neoplasm Proteins - chemistry; Neoplasm Proteins - genetics; Neoplasm Proteins - metabolism; Protein Binding; Protein Conformation; Protein-RNA interaction; RNA Cap Analogs - chemistry; RNA Cap Analogs - metabolism; RNA Cap-Binding Proteins - chemistry; RNA Cap-Binding Proteins - genetics; RNA Cap-Binding Proteins - metabolism; RNA Caps - chemistry; RNA Caps - genetics; RNA Caps - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - metabolism; Spectrometry, Fluorescence; Static Electricity; Thermodynamics; Titration assay; Titration assay; IFIT; Fluorescence probe; Protein-RNA interaction
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2020.09.006

The interferon-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding proteins that are very highly expressed during antiviral response of immune system. IFIT proteins recognize and tightly bind foreign RNA particles. These are primarily viral RNAs ended with triphosphate at the 5’ or lacking methylation of the first cap-proximal nucleotide but also in vitro transcribed RNA synthesized in the laboratory. Recognition of RNA by IFIT proteins leads to the formation of stable RNA/IFIT complexes and translational shut off of non-self transcripts. Here, we present a fluorescent-based assay to study the interaction between RNA molecules and IFIT family proteins. We have particularly focused on two representatives of this family: IFIT1 and IFIT5. We found a probe that competitively with RNA binds the positively charged tunnel in these IFIT proteins. The use of this probe for IFIT titration allowed us to evaluate the differences in binding affinities of mRNAs with different variants of 5’ ends. •Fluorescence-based assay is a fast and easy tool for detection of RNA-IFIT binding.•Bis-ANS probe demonstrates effective binding to the IFIT proteins.•Bis-ANS is located inside the RNA binding tunnel of the IFIT proteins.•Developed assay enables binding studies at low RNA concentrations.