Quantitative analysis of biomolecular condensates on a modified support

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Abstract

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. 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 substrate, chemically polished glass was treated with 3-aminopropyltriethoxysilane in ethyl alcohol at pH 4.5–5.5. To obtain the DSC-APTES substrate, the APTES substrate was additionally functionalized by treating with N,N′-disuccinimidyl carbonate in the presence of diisopropylethylamine in anhydrous acetone. 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. 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. 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.

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About the authors

A. S. Shtork

Federal Research and Clinical Center of Physical-Chemical Medicine

Author for correspondence.
Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

Iu. I. Pavlova

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

J. I. Svetlova

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

M. S. Iudin

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

E. N. Grafskaia

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

V. A. Manuvera

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

S. E. Alieva

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

A. M. Varizhuk

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

V. N. Lazarev V. N. Lazarev V. N. Lazarev

Federal Research and Clinical Center of Physical-Chemical Medicine

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow

T. S. Vedekhina

Federal Research and Clinical Center of Physical-Chemical Medicine; MIREA — Russian technological university

Email: vedekhina.ts@rcpcm.ru
Russian Federation, Moscow; Moscow

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Supplementary files

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2. Fig. 1. Formation of biocondensates and their interactions with substrates. The upper panel schematically shows the formation of condensates based on peptides or proteins with unstructured domains. The lower panel shows probable contacts of biopolymer fragments exposed on the condensate surface with the surface of a glass substrate or a substrate treated with 3-aminopropyltriethoxysilane (APTES), as well as a substrate obtained by functionalization of APTES with N,N'-disuccinimidyl carbonate (DSC) in the presence of diisopropylethylamine (DIEA). Upon contact with an unmodified glass substrate, hydrogen bonds and van der Waals interactions presumably play a key role. The APTES substrate is capable of Coulomb interactions with phosphates of the RNA backbone. The DSC-APTES substrate carries an activated carboxyl group on the surface for covalent attachment of proteins to lysine residues with the formation of the corresponding amide. The by-product of functionalization (the result of the interaction of DSC with two neighboring APTES residues), as well as the DSC-APTES hydrolysis product that accumulates during incubation in aqueous solutions, are capable of additionally fixing condensates due to weak non-covalent interactions.

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3. Fig. 2. Analysis of the effect of substrate modification on the mobility of condensates. (a) – Visualization of condensates on different substrates by fluorescence microscopy immediately after application (upper row) and after 5 min of incubation (lower row). Left panel: condensates of SR peptide (10 mg/ml, 1% FITC-labeled) with RNA (10 mg/ml) in 10 mM sodium phosphate buffer (pH 7.4). Right panel: condensates of N protein (50 μM, 15% RED-labeled) with RNA (0.5 mg/ml) in 50 mM sodium phosphate buffer (pH 7) with the addition of 50 mM NaCl; (b) – examples of images obtained at 30 s intervals for the analysis of condensate mobility; (c) – results of semi-automated analysis of condensate mobility. The analysis included calculation of the root-mean-square displacement of the condensate for 10 s based on a trajectory of 3–5 frames taken at 10-second intervals, with visual control of trajectory construction. A generalization of data for 10 trajectories on each type of substrate is presented. * p < 0.05; ** p < 0.01 (Student's test, Mann–Whitney test); (d) – results of automatic analysis of condensate mobility. Histograms of the distribution of the root-mean-square deviation were constructed for 100 trajectories for each type of substrate without visual control of trajectory construction.

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4. Fig. 3. Analysis of the influence of the substrate on the ratio of condensate components and the phase state of the system. (a) Examples of visualization of condensates of N-protein (50 μM, 15% RED-labeled) with RNA (0.5 mg/ml, 15% FAM-labeled LNA oligonucleotide) in a sodium phosphate buffer solution (pH 7) based on protein fluorescence (in the red channel) and LNA fluorescence (in the green channel); (b) assessment of protein and LNA colocalization in condensates based on fluorescence microscopy data (three replicates for each type of substrate). Quantitative analysis included calculation of the percentage of droplets visualized in both channels (method 1) and determination of the Pearson correlation coefficient, reflecting the correlation of fluorescence intensities in the red and green channels (method 2). * p < 0.05 (Student's test); (c) Examples of visualization of condensate dissolution and gel transition induced by the addition of 10% 1,6-hexanediol (HD) and a perylene-based ligand (Peryl-3b) to a final concentration of 20 μM, on different substrates.

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5. Fig. 4. Analysis of the influence of the substrate on the observed area of ​​condensates and the obtained phase diagrams. (a) – Comparison of the normalized total area of ​​condensates per unit area of ​​the coverslip at different concentrations of protein and RNA, calculated in the Droplet_Calc program for three series of images: on glass (gray), on an APTES substrate (orange) or on DSC-APTES (green). The values ​​are normalized to the maximum for each series, the results (fragments of the phase diagrams) are presented in the format of heat maps; (b) – example images corresponding to the diagonal elements of the heat maps; (c) – non-normalized values ​​of the total area of ​​condensates corresponding to the diagonal elements of the heat maps in panel (a) and the images in panel (b).

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