Quantitative Analysis of Biomolecular Condensates on a Modified Support

Abstract

Objective: Biomolecular condensates are associates of biopolymers formed in aqueous solutions via “liquid-liquid” phase separation. Aberrant phase transitions of proteins or nucleic acids underlie several pathologies, and the need for their in vitro models stimulates the development of methods for biocondensate investigation. This work addresses the key problem of visualizing labeled protein-RNA condensates using fluorescence microscopy. Methods: The SARS-CoV-2 N-protein with a C-terminal hexahistidine tag was expressed in Escherichia coli BL21-Gold(DE3) and isolated by metal chelate chromatography. The N-protein was labeled with the RED dye, which emits fluorescence in the far-red range of the spectrum, using the RED-NHS dye. Commercially available RNA isolated from Torula yeast was used as random RNA to obtain condensates with the N-protein and SR-rich peptide. In experiments to test the colocalization of the condensate components, a labeled modified oligonucleotide forming an SL4 hairpin with an elongated stem was added to the random RNA. To obtain the APTES support, chemically polished glass was treated with 3-aminopropyltriethoxysilane in ethyl alcohol at pH 4.5–5.5. To obtain the DSC-APTES support, the APTES support was additionally functionalized by treating with N,N′-disuccinimidyl carbonate in the presence of diisopropylethylamine in anhydrous acetone. A quantitative assessment of condensate formation was performed using fluorescence microscopy data. The FastTrack program was used to assess droplet mobility. The Droplet_Calc program was used to assess the droplet area and curvature coefficient. Results and Discussion: The mobility of the condensates in a sample layer on glass complicates data processing. In previous studies, condensate immobilization on 3-aminopropyltriethoxysilane-treated glass (APTES), was proposed to overcome this problem. The APTES support allows non-covalent RNA/DNA binding but is suboptimal for proteins. By treating APTES with N,N′-disuccinimidyl carbonate, we obtained an alternative support, DSC-APTES, which allows covalent binding of protein fragments via lysine residues. A comparative analysis of known condensates on the abovementioned supports revealed their decreased mobility on APTES/DSC-APTES, and the optimal type of support modification depended on the condensate composition. Condensate immobilization improved image quality and increased the colocalization of the oligonucleotide and protein components. It also facilitated the quantitative analysis of the phase separation based on the condensate fractions. New software, Droplet_Calc, was developed to automate condensate identification and fraction calculation. The results confirmed the advantages of APTES and DSC-APTES over glass when analyzing the concentration dependence of the condensate fraction and creating phase diagrams. Conclusions: Thus, the optimization of the support and the automation of image processing pave the way for rapid and reliable quantitative analysis of biopolymer phase transitions, which may find application in the screening of therapeutic agents disrupting pathogenic condensates.